Compounds, compositions, and methods

ABSTRACT

Compounds useful for treating cellular proliferative diseases and disorders by modulating the activity of KSP are disclosed.

This application claims the benefit of U.S. Patent Application No.60/528,219, filed Dec. 8, 2003, which is hereby incorporated byreference.

This invention relates to compounds which are inhibitors of the mitotickinesin KSP and are useful in the treatment of cellular proliferativediseases, for example cancer, hyperplasias, restenosis, cardiachypertrophy, immune disorders, fungal disorders, and inflammation.

Among the therapeutic agents used to treat cancer are the taxanes andvinca alkaloids, which act on microtubules. Microtubules are the primarystructural element of the mitotic spindle. The mitotic spindle isresponsible for distribution of replicate copies of the genome to eachof the two daughter cells that result from cell division. It is presumedthat disruption of the mitotic spindle by these drugs results ininhibition of cancer cell division, and induction of cancer cell death.However, microtubules form other types of cellular structures, includingtracks for intracellular transport in nerve processes. Because theseagents do not specifically target mitotic spindles, they have sideeffects that limit their usefulness.

Improvements in the specificity of agents used to treat cancer is ofconsiderable interest because of the therapeutic benefits which would berealized if the side effects associated with the administration of theseagents could be reduced. Traditionally, dramatic improvements in thetreatment of cancer are associated with identification of therapeuticagents acting through novel mechanisms. Examples of this include notonly the taxanes, but also the camptothecin class of topoisomerase Iinhibitors. From both of these perspectives, mitotic kinesins areattractive targets for new anti-cancer agents.

Mitotic kinesins are enzymes essential for assembly and function of themitotic spindle, but are not generally part of other microtubulestructures, such as in nerve processes. Mitotic kinesins play essentialroles during all phases of mitosis. These enzymes are “molecular motors”that transform energy released by hydrolysis of ATP into mechanicalforce which drives the directional movement of cellular cargoes alongmicrotubules. The catalytic domain sufficient for this task is a compactstructure of approximately 340 amino acids. During mitosis, kinesinsorganize microtubules into the bipolar structure that is the mitoticspindle. Kinesins mediate movement of chromosomes along spindlemicrotubules, as well as structural changes in the mitotic spindleassociated with specific phases of mitosis. Experimental perturbation ofmitotic kinesin function causes malformation or dysfunction of themitotic spindle, frequently resulting in cell cycle arrest and celldeath.

Among the mitotic kinesins which have been identified is KSP. KSPbelongs to an evolutionarily conserved kinesin subfamily of plusend-directed microtubule motors that assemble into bipolar homotetramersconsisting of antiparallel homodimers. During mitosis KSP associateswith microtubules of the mitotic spindle. Microinjection of antibodiesdirected against KSP into human cells prevents spindle pole separationduring prometaphase, giving rise to monopolar spindles and causingmitotic arrest and induction of programmed cell death. KSP and relatedkinesins in other, non-human, organisms, bundle antiparallelmicrotubules and slide them relative to one another, thus forcing thetwo spindle poles apart. KSP may also mediate in anaphase B spindleelongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEg5) has been described (Blangy, et al., Cell,83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42(1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, etal., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell MotilCytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci., 111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBankaccession numbers: X85137, NM004523 and U37426), and a fragment of theKSP gene (TRIP5) has been described (Lee, et al., Mol Endocrinol.,9:243-54 (1995); GenBank accession number L40372). Xenopus KSP homologs(Eg5), as well as Drosophila KLP61 F/KRP1 30 have been reported.

Mitotic kinesins, including KSP, are attractive targets for thediscovery and development of novel antimitotic chemotherapeutics.Accordingly, it is an object of the present invention to providecompounds, compositions and methods useful in the inhibition of KSP.

In accordance with the objects outlined above, the present inventionprovides compounds that can be used to treat cellular proliferativediseases. The compounds are KSP inhibitors, such as human KSPinhibitors. The present invention also provides compositions comprisingsuch compounds, and methods utilizing such compounds or compositions,which can be used to treat cellular proliferative diseases

In one aspect, the invention relates to methods for treating cellularproliferative diseases, and for treating disorders by inhibiting theactivity of KSP. The methods employ at least one chemical entity chosenfrom compounds of Formula I:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein,

-   -   W, X, Y, and Z are independently —N═, N, —C═, CR₁₈, O or S and Z        is optionally absent, provided that:        -   the ring comprising W, X, Y, and optionally Z is            heterocyclic or heteroaromatic;        -   no more than two of W, X, Y, and Z is —N═, and        -   W, X, or Y can be O or S only when Z is absent;    -   the dashed lines in the structure depict optional double bonds;    -   T and T′ are independently a covalent bond or optionally        substituted lower alkylene;    -   R₁ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-, optionally substituted aralkyl-,        optionally substituted heteroaryl-, and optionally substituted        heteroaralkyl-;    -   R₂ and R_(2′) are independently chosen from hydrogen, optionally        substituted alkyl-, optionally substituted aryl-, optionally        substituted aralkyl-, optionally substituted heteroaryl-, and        optionally substituted heteroaralkyl-; or R₂ and R_(2′) taken        together form an optionally substituted 3- to 7-membered ring;    -   R₁₂ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-, optionally substituted aralkyl-,        optionally substituted heteroaryl-, optionally substituted        heteroaralkyl-, —C(O)—R₃, and —S(O)₂—R_(3a);    -   R₃ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-, optionally substituted aralkyl-,        optionally substituted heteroaryl-, optionally substituted        heteroaralkyl-, R₁₅O— and R₁₇—NH—;    -   R_(3a) is chosen from optionally substituted alkyl-, optionally        substituted aryl-, optionally substituted aralkyl-, optionally        substituted heteroaryl-, optionally substituted heteroaralkyl-,        and R₁₇—NH—;    -   R₄ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-, optionally substituted aralkyl-,        optionally substituted heteroaralkyl-, and optionally        substituted heterocyclyl-;    -   or R₄ taken together with R₁₂, and the nitrogen to which they        are bound, form an optionally substituted 5- to 12-membered        nitrogen-containing heterocycle, which optionally incorporates        one or two additional heteroatoms, selected from N, O, and S in        the heterocycle ring;    -   or R₄ taken together with R₂ form an optionally substituted 5-        to 12-membered nitrogen-containing heterocycle, which optionally        incorporates one or two additional heteroatoms, selected from N,        O, and S in the heterocycle ring;    -   R₅, R₆, R₇, R₈, and R₁₈ are independently chosen from hydrogen,        acyl, optionally substituted alkyl-, optionally substituted        alkoxy, halogen, hydroxyl, nitro, cyano, optionally substituted        amino, alkylsulfonyl-, alkylsulfonamido-, alkylthio-,        carboxyalkyl-, carboxamido-, aminocarbonyl-, optionally        substituted aryl and optionally substituted heteroaryl-,        provided that R₅, R₆, R₇ and R₈ is absent where W, X, Y, or Z,        respectively, is —N═, O, S or absent;    -   R₁₅ is chosen from optionally substituted alkyl-, optionally        substituted aryl-, optionally substituted aralkyl-, optionally        substituted heteroaryl-, and optionally substituted        heteroaralkyl-; and    -   R₁₇ is hydrogen, optionally substituted alkyl, optionally        substituted aryl, optionally substituted aralkyl, optionally        substituted heteroaryl, or optionally substituted        heteroaralkyl-.

In some embodiments, the methods employ at least one chemical entitychosen from compounds of Formula II:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein R₁, R₂, R₂′, R₄ through R₈, R₁₂, T, and T′ are as defined aboveand provided that T and T′ are not both absent.

In some embodiments, the methods employ at least one chemical entitychosen from compounds of Formula III:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein

-   -   R₁, R₃, R_(3a), R₁₅, R₁₇ and R₅ through R₈ are as defined above;        and    -   either R₂ and R_(2′) are independently chosen from hydrogen,        optionally substituted alkyl-, optionally substituted aryl-,        optionally substituted aralkyl-, optionally substituted        heteroaryl-, and optionally substituted heteroaralkyl- or R₂ and        R_(2′) taken together form an optionally substituted 3- to        7-membered ring; and R₁₂ taken together with R₄, and the        nitrogen to which they are bound, form an optionally substituted        5- to 12-membered nitrogen-containing heterocycle, which        optionally incorporates one or two additional heteroatoms,        selected from N, O, and S in the heterocycle ring, provided that        such 5-membered nitrogen-containing heterocycle is not an        optionally substituted imidazolyl or imidazolinyl ring;    -   or R₂ taken together with R₄ form an optionally substituted 5-        to 12-membered nitrogen-containing heterocycle, which optionally        incorporates one or two additional heteroatoms, selected from N,        O, and S in the heterocycle ring; R_(2′) is hydrogen, optionally        substituted alkyl-, optionally substituted aryl-, optionally        substituted aralkyl-, optionally substituted heteroaryl-, or        optionally substituted heteroaralkyl; and R₁₂ is chosen from        hydrogen, optionally substituted alkyl-, optionally substituted        aryl-, optionally substituted aralkyl-, optionally substituted        heteroaryl-, optionally substituted heteroaralkyl-, —C(O)—R₃,        and —S(O)₂—R_(3a).

In one aspect, the invention relates to methods for treating cellularproliferative diseases and other disorders that can be treated byinhibiting KSP by the administration of a therapeutically effectiveamount of at least one chemical entity chosen from compounds of FormulaI, II, and III and pharmaceutically acceptable salts, solvates, crystalforms, chelates, non-covalent complexes, prodrugs, and mixtures thereof.Such diseases and disorders include cancer, hyperplasia, restenosis,cardiac hypertrophy, immune disorders, fungal disorders andinflammation. It will be understood that the method can employ one ormore of the foregoing compounds.

In another aspect, the invention relates to at least one chemical entitychosen from compounds of Formula I, II, and III and pharmaceuticallyacceptable salts, solvates, crystal forms, chelates, non-covalentcomplexes, prodrugs, and mixtures thereof. The invention also relates topharmaceutical compositions comprising: a therapeutically effectiveamount of at least one chemical entity chosen from compounds of FormulaI, II, and III and pharmaceutically acceptable salts, solvates, crystalforms, chelates, non-covalent complexes, prodrugs, and mixtures thereofand at least one pharmaceutical excipient. In another aspect, thecomposition further comprises a chemotherapeutic agent other than achemical entity of the present invention.

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise. The following abbreviations and terms have the indicatedmeanings throughout:

-   -   Ac=acetyl    -   BNB=4-bromomethyl-3-nitrobenzoic acid    -   Boc=t-butyloxy carbonyl    -   Bu=butyl    -   c-=cyclo    -   CBZ =carbobenzoxy=benzyloxycarbonyl    -   DBU=diazabicyclo[5.4.0]undec-7-ene    -   DCM=dichloromethane=methylene chloride=CH₂Cl₂    -   DCE=dichloroethane    -   DEAD=diethyl azodicarboxylate    -   DIC=diisopropylcarbodiimide    -   DIEA=N,N-diisopropylethylamine    -   DMAP=4-N,N-dimethylaminopyridine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   DVB=1,4-divinylbenzene    -   EEDQ=2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline    -   Et=ethyl    -   Fmoc=9-fluorenylmethoxycarbonyl    -   GC=gas chromatography    -   HATU=O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HMDS=hexamethyldisilazane    -   HOAc=acetic acid    -   HOBt=hydroxybenzotriazole    -   Me=methyl    -   mesyl=methanesulfonyl    -   MTBE=methyl t-butyl ether    -   NMO=N-methylmorpholine oxide    -   PEG=polyethylene glycol    -   Ph=phenyl    -   PhOH=phenol    -   PfP=pentafluorophenol    -   Pht=phthalyl    -   PPTS=pyridinium p-toluenesulfonate    -   Py=pyridine    -   PyBroP=bromo-tris-pyrrolidino-phosphonium hexafluorophosphate    -   rt=room temperature    -   sat'd=saturated    -   s-=secondary    -   t-=tertiary    -   TBDMS=t-butyldimethylsilyl    -   TES=triethylsilyl    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   TMOF=trimethyl orthoformate    -   TMS=trimethylsilyl    -   tosyl=p-toluenesulfonyl    -   Trt=triphenylmethyl

Alkyl is intended to include linear, branched, or cyclic aliphatichydrocarbon structures and combinations thereof, which structures may besaturated or unsaturated. Lower-alkyl refers to alkyl groups of from 1to 5 carbon atoms, such as from 1 to 4 carbon atoms. Examples oflower-alkyl groups include methyl-, ethyl-, propyl-, isopropyl-, butyl-,s- and t-butyl and the like. In some embodiments, alkyl groups are thoseof C₂₀ or below. In some embodiments, alkyl groups are those of C₁₃ orbelow. Cycloalkyl is a subset of alkyl and includes cyclic aliphatichydrocarbon groups of from 3 to 13 carbon atoms. Examples of cycloalkylgroups include c- propyl-, c-butyl-, c-pentyl-, norbornyl-, adamantyland the like. Cycloalkyl-alkyl- is another subset of alkyl and refers tocycloalkyl attached to the parent structure through a non-cyclic alkyl-.Examples of cycloalkyl-alkyl- include cyclohexylmethyl-,cyclopropylmethyl-, cyclohexylpropyl-, and the like. In thisapplication, alkyl includes alkanyl-, alkenyl and alkynyl residues; itis intended to include vinyl-, allyl-, isoprenyl and the like. When analkyl residue having a specific number of carbons is named, allgeometric isomers having that number of carbons are intended to beencompassed; thus, for example, “butyl” is meant to include n-butyl-,sec-butyl-, isobutyl and t-butyl-; “propyl” includes n-propyl-,isopropyl-, and c-propyl-.

Alkylene-, alkenylene-, and alkynylene- are other subsets of alkyl-,including the same residues as alkyl-, but having two points ofattachment within a chemical structure. Examples of alkylene includeethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), dimethylpropylene(—CH₂C(CH₃)₂CH₂—) and cyclohexylpropylene (—CH₂CH₂CH(C₆H₁₃)—). Likewise,examples of alkenylene include ethenylene (—CH═CH—), propenylene(—CH═CH—CH₂—), and cyclohexylpropenylene (—CH═CHCH(C₆H₁₃)—). Examples ofalkynylene include ethynylene (—C≡C—) and propynylene (—CH≡CH—CH₂—).

Cycloalkenyl is a subset of alkyl and includes unsaturated cyclichydrocarbon groups of from 3 to 13 carbon atoms. Examples ofcycloalkenyl groups include c-hexenyl-, c-pentenyl and the like.

Alkoxy or alkoxyl refers to an alkyl group, such as from 1 to 8 carbonatoms, of a straight, branched, or cyclic configuration, or acombination thereof, attached to the parent structure through an oxygen(i.e., the group alkyl-O—). Examples include methoxy-, ethoxy-,propoxy-, isopropoxy-, cyclopropyloxy-, cyclohexyloxy- and the like.Lower-alkoxy refers to alkoxy groups containing one to four carbons.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight,branched, or cyclic configuration or a combination thereof, attached tothe parent structure through a carbonyl functionality. Such groups maybe saturated or unsaturated, and aliphatic or aromatic. One or morecarbons in the acyl residue may be replaced by oxygen, nitrogen (e.g.,carboxamido), or sulfur as long as the point of attachment to the parentremains at the carbonyl. Examples include acetyl-, benzoyl-, propionyl-,isobutyryl-, oxalyl-, t-butoxycarbonyl-, benzyloxycarbonyl,morpholinylcarbonyl, and the like. Lower-acyl refers to acyl groupscontaining one to four carbons.

Amino refers to the group —NH₂. The term “substituted amino” refers tothe group —NHR or —NRR where each R is independently selected from thegroup: optionally substituted alkyl-, optionally substituted alkoxy,optionally substituted aminocarbonyl-, optionally substituted aryl-,optionally substituted heteroaryl-, optionally substitutedheterocyclyl-, acyl-, alkoxycarbonyl-, sulfanyl-, sulfinyl andsulfonyl-, e.g., diethylamino, methylsulfonylamino,furanyl-oxy-sulfonamino. Substituted amino includes the group—NR^(c)COR^(b), —NR^(c)CO₂R^(a), or —NR^(c)CONR^(b)R^(c), where

-   -   R^(a) is optionally substituted C₁-C₆ alkyl-, aryl-,        heteroaryl-, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl-        group;    -   R^(b) is H or optionally substituted C₁-C₆ alkyl-, aryl-,        heteroaryl-, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl-        group; and    -   R^(c) is hydrogen, alkyl-; aryl- or heteroaryl-;        where each optionally substituted R^(b) group is independently        unsubstituted or substituted with one or more substituents        independently selected from C₁-C₄ alkyl-, aryl-, heterocyclyl-,        aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl-,        —OC₁—C₄ alkyl, —OC₁—C₄ alkylphenyl, —C₁-C₄ alkyl-OH, —OC₁—C₄        haloalkyl, halogen, —OH, —NH₂, —NR^(c)C(NR^(b))(NR^(b)R^(c))        (i.e, guanidine), —NR^(c)CR^(b)NR^(b)R^(c), —CNR^(c)NR^(b)R^(c),        —C₁-C₄ alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄        alkyl), —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄        alkylphenyl), cyano, nitro, oxo (as a substitutent for        heteroaryl), —CO₂H, —C(O)OC₁—C₄ alkyl-, —CON(C₁-C₄ alkyl)(C₁-C₄        alkyl), —CONH(C₁-C₄ alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl),        —NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄        alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl-, —C(O)C₁-C₄ phenyl-,        —C(O)C₁-C₄ haloalkyl-, —OC(O)C₁—C₄ alkyl-, —SO₂(C₁-C₄ alkyl),        —SO₂(phenyl), —SO₂(C₁-C₄ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₄        alkyl), —SO₂NH(phenyl), —NHSO₂(C₁-C₄ alkyl), —NHSO₂(phenyl), and        —NHSO₂(C₁-C₄ haloalkyl).

Antimitotic refers to a drug for inhibiting or preventing mitosis, forexample, by causing metaphase arrest. Some antitumour drugs blockproliferation and are considered antimitotics.

Aryl encompasses:

-   -   5- and 6-membered carbocyclic aromatic rings, for example,        benzene;    -   bicyclic ring systems wherein at least one ring is carbocyclic        and aromatic, for example, naphthalene, indane, and tetralin;        and    -   tricyclic ring systems wherein at least one ring is carbocyclic        and aromatic, for example, fluorene.        For example, aryl includes 5- and 6-membered carbocyclic        aromatic rings fused to a 5- to 7-membered heterocycloalkyl ring        containing 1 or more heteroatoms chosen from N, O, and S.        Bivalent radicals formed from substituted benzene derivatives        and having the free valences at ring atoms are named as        substituted phenylene radicals. Bivalent radicals derived from        univalent polycyclic hydrocarbon radicals whose names end in        “-yl” by removal of one hydrogen atom from the carbon atom with        the free valence are named by adding “-idene” to the name of the        corresponding univalent radical, e.g., a naphthyl group with two        points of attachment is termed naphthylidene.

“Heteroaryl” encompasses:

-   -   5- to 7-membered aromatic, monocyclic rings containing one or        more, for example, from 1 to 4, or in certain embodiments, from        1 to 3, heteroatoms chosen from N, O and S, with the remaining        ring atoms being carbon; and    -   bicyclic heterocyclic rings containing one or more, for example,        from 1 to 4, or in certain embodiments, from 1 to 3, heteroatoms        chosen from N, O, and S, with the remaining ring atoms being        carbon and wherein at least one heteroatom is present in an        aromatic ring.        For example, heteroaryl includes a 5- to 7-membered        heterocycloalkyl, aromatic ring fused to a 5- to 7-membered        cycloalkyl ring. For such fused, bicyclic heteroaryl ring        systems wherein only one of the rings contains one or more        heteroatoms, the point of attachment may be at the        heteroaromatic ring or the cycloalkyl ring. When the total        number of S and O atoms in the heteroaryl group exceeds 1, those        heteroatoms are not adjacent to one another. In certain        embodiments, the total number of S and O atoms in the heteroaryl        group is not more than 2. In certain embodiments, the total        number of S and O atoms in the aromatic heterocycle is not more        than 1. Examples of heteroaryl groups include, but are not        limited to, systems (as numbered from the linkage position        assigned priority 1), such as 2-pyridyl, 3-pyridyl, 4-pyridyl,        2,3-pyrazinyl, 3,4-pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl,        2,3-pyrazolinyl, 2,4-imidazolinyl, isoxazolinyl, oxazolinyl,        thiazolinyl, thiadiazolinyl, tetrazolyl, thienyl,        benzothiophenyl, furanyl, benzofuranyl, benzoimidazolinyl,        indolinyl, pyridizinyl, triazolyl, quinolinyl, pyrazolyl, and        5,6,7,8-tetrahydroisoquinoline. Bivalent radicals derived from        univalent heteroaryl radicals whose names end in “-yl” by        removal of one hydrogen atom from the atom with the free valence        are named by adding “-idene” to the name of the corresponding        univalent radical, e.g., a pyridyl group with two points of        attachment is a pyridylidene.

Aralkyl- refers to a residue in which an aryl moiety is attached to theparent structure via an alkyl residue. Examples include benzyl-,phenethyl-, phenylvinyl-, phenylallyl and the like. Heteroaralkyl-refers to a residue in which a heteroaryl moiety is attached to theparent structure via an alkyl residue. Examples include furanylmethyl-,pyridinylmethyl-, pyrimidinylethyl and the like.

Aralkoxy- refers to the group —O-aralkyl. Similarly, heteroaralkoxy-refers to the group —O-heteroaralkyl-; aryloxy- refers to the group—O-aryl-; acyloxy- refers to the group —O-acyl-; heteroaryloxy- refersto the group —O-heteroaryl-; and heterocyclyloxy- refers to the group—O-heterocyclyl (i.e., aralkyl-, heteroaralkyl-, aryl-, acyl-,heterocyclyl-, or heteroaryl is attached to the parent structure throughan oxygen).

Carboxyalkyl- refers to the group -alkyl-COOH.

Aminocarbonyl refers to the group —CONR^(b)R^(c), where

-   -   R^(b) is H or optionally substituted C₁-C₆ alkyl-, aryl-,        heteroaryl-, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl-        group; and    -   R^(c) is hydrogen, alkyl-; aryl- or heteroaryl-; and    -   where each optionally substituted R^(b) group is independently        unsubstituted or substituted with one or more substituents        independently selected from C₁-C₄ alkyl-, aryl-, heterocyclyl-,        aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl-,        —OC₁—C₄ alkyl-, —OC₁—C₄ alkylphenyl-, —C₁-C₄ alkyl-OH, —OC₁—C₄        haloalkyl-, halogen, —OH, —NH₂, —C₁-C₄ alkyl-NH₂, —N(C₁-C₄        alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)(C₁-C₄        alkylphenyl), —NH(C₁-C₄ alkylphenyl), cyano, nitro, oxo (as a        substitutent for heteroaryl), —CO₂H, —C(O)OC₁—C₄ alkyl-,        —CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), —CONH(C₁-C₄ alkyl), —CONH₂,        —NHC(O)(C₁-C₄ alkyl), —NHC(O)(phenyl), —N(C₁-C₄ alkyl)C(O)(C₁-C₄        alkyl), —N(C₁-C₄ alkyl)C(O)(phenyl), —C(O)C₁-C₄ alkyl-,        —C(O)C₁-C₄ phenyl-, —C(O)C₁-C₄ haloalkyl-, —OC(O)C₁—C₄ alkyl-,        —SO₂(C₁-C₄ alkyl), —SO₂(phenyl), —SO₂(C₁-C₄ haloalkyl), —SO₂NH₂,        —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl), —NHSO₂(C₁-C₄ alkyl),        —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl). Aminocarbonyl is        meant to include carbamoyl-; lower-alkyl carbamoyl-;        benzylcarbamoyl-; phenylcarbamoyl-; methoxymethyl-carbamoyl-;        and the like.

Halogen or halo refers to fluorine, chlorine, bromine or iodine.Fluorine, chlorine and bromine are preferred. Dihaloaryl-, dihaloalkyl-,trihaloaryl etc. refer to aryl and alkyl substituted with the designatedplurality of halogens (here, 2, 2 and 3, respectively), but notnecessarily a plurality of the same halogen; thus4-chloro-3-fluorophenyl is within the scope of dihaloaryl-.

By “heterocycloalkyl” or “heterocyclyl” is meant a single aliphatic ringcontaining at least 2 carbon atoms in addition to 1-3 heteroatomsindependently selected from oxygen, sulfur, and nitrogen, as well ascombinations comprising at least one of the foregoing heteroatoms.Suitable heterocycloalkyl groups include, for example (as numbered fromthe linkage position assigned priority 1), 2-pyrrolinyl,2,4-imidazolidinyl, 2,3-pyrazolidinyl, 2-piperidyl, 3-piperidyl,4-piperdyl, and 2,5-piperzinyl. Morpholinyl groups are alsocontemplated, including 2-morpholinyl and 3-morpholinyl (numberedwherein the oxygen is assigned priority 1).

A leaving group or atom is any group or atom that will, under thereaction conditions, cleave from the starting material, thus promotingreaction at a specified site. Suitable examples of such groups unlessotherwise specified are halogen atoms, mesyloxy,p-nitrobenzensulphonyloxy and tosyloxy groups.

Optional or optionally means that the subsequently described event orcircumstance may or may not occur, and that the description includesinstances where said event or circumstances occurs and instances inwhich it does not. For example, “optionally substituted alkyl” includes“alkyl” and “substituted alkyl” as defined herein. It will be understoodby those skilled in the art with respect to any group containing one ormore substituents that such groups are not intended to introduce anysubstitution or substitution patterns that are sterically impracticaland/or synthetically non-feasible and/or inherently unstable.

Substituted alkoxy refers to alkoxy wherein the alkyl constituent issubstituted (i.e., —O-(substituted alkyl)). One suitable substitutedalkoxy group is “polyalkoxy” or —O-(optionally substitutedalkylene)-(optionally substituted alkoxy), and includes groups such as—OCH₂CH₂OCH₃, and residues of glycol ethers such as polyethyleneglycol,and —O(CH₂CH₂O)_(x)CH₃, where x is an integer of about 2-20, such asabout 2-10, for example, about 2-5. Another suitable substituted alkoxygroup is hydroxyalkoxy or —OCH₂(CH₂)_(y)OH, where y is an integer ofabout 1-10, such as about 1-4.

Substituted- alkyl-, aryl-, and heteroaryl- refer respectively toalkyl-, aryl-, and heteroaryl wherein one or more (up to about 5, suchas up to about 3) hydrogen atoms are replaced by a substituentindependently selected from the group: —R^(a), —OR^(b), —O(C₁-C₂alkyl)O— (as an aryl substituent), —SR^(b), —NR^(b)R^(c),—NR^(c)C(NR^(b))(NR^(b)R^(c)) (i.e, guanidine),—NR^(c)CR^(b)NR^(b)R^(c), —CNR^(c)NR^(b)R^(c), halogen, cyano, nitro,—COR^(b), —CO₂R^(b), —CONR^(b)R^(c), —OCOR^(b), —OCO₂R^(a),—OCONR^(b)R^(c), —NR^(c)COR^(b), —NR^(c)CO₂R^(a), —NR^(c)CONR^(b)R^(c),—SOR^(a), —SO₂R^(a), —SO₂NR^(b)R^(c), and —NR^(c)SO₂R^(a),

-   -   where R^(a) is an optionally substituted C₁-C₆ alkyl-, aryl-,        heteroaryl-, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl-        group,    -   R^(b) is H or optionally substituted C₁-C₆ alkyl-, aryl-,        heteroaryl-, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl-        group;    -   R^(c) is hydrogen, alkyl-; aryl- or heteroaryl-; and        where each optionally substituted R^(a) group and R^(b) group is        independently unsubstituted or substituted with one or more        substituents independently selected from C₁-C₄ alkyl-, aryl-,        heterocyclyl-, aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄        haloalkyl-, —OC₁—C₄ alkyl-, —OC₁—C₄ alkylphenyl-, —C₁-C₄        alkyl-OH, —OC₁—C₄ haloalkyl-, halogen, —OH, —NH₂, —C₁-C₄        alkyl-NH₂, —N(C₁-C₄ alkyl)(C₁-C₄ alkyl), —NH(C₁-C₄ alkyl),        —N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), —NH(C₁-C₄ alkylphenyl),        cyano, nitro, oxo (as a substitutent for heteroaryl), —CO₂H,        —C(O)OC₁—C₄ alkyl-, —CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), —CONH(C₁-C₄        alkyl), —CONH₂, —NHC(O)(C₁-C₄ alkyl), —NHC(O)(phenyl), —N(C₁-C₄        alkyl)C(O)(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)C(O)(phenyl), —C(O)C₁-C₄        alkyl-, —C(O)C₁-C₄ phenyl-, —C(O)C₁-C₄ haloalkyl-, —OC(O)C₁—C₄        alkyl-, —SO₂(C₁-C₄ alkyl), —SO₂(phenyl), —SO₂(C₁-C₄ haloalkyl),        —SO₂NH₂, —SO₂NH(C₁-C₄ alkyl), —SO₂NH(phenyl), —NHSO₂(C₁-C₄        alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₄ haloalkyl).

Sulfanyl refers to the groups: —S-(optionally substituted alkyl),—S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl), and —S-(optionally substituted heterocyclyl).

Sulfinyl refers to the groups: —S(O)—H, —S(O)-(optionally substitutedalkyl), —S(O)-optionally substituted aryl), —S(O)-(optionallysubstituted heteroaryl), —S(O)-(optionally substituted heterocyclyl);and —S(O)-(optionally substituted amino).

Sulfonyl refers to the groups: —S(O₂)—H, —S(O₂)-(optionally substitutedalkyl), —S(O₂)-optionally substituted aryl), —S(O₂)-(optionallysubstituted heteroaryl), —S(O₂)-(optionally substituted heterocyclyl),—S(O₂)-(optionally substituted alkoxy), —S(O₂)-optionally substitutedaryloxy), —S(O₂)-(optionally substituted heteroaryloxy),—S(O₂)-(optionally substituted heterocyclyloxy); and —S(O₂)-(optionallysubstituted amino).

Pharmaceutically acceptable salts refers to those salts that retain thebiological effectiveness of the free compound and that are notbiologically undesirable or unsuitable for pharmaceutical use, formedwith a suitable acid or base, and includes pharmaceutically acceptableacid addition salts and base addition salts. Pharmaceutically acceptableacid addition salts include those derived from inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and those derived from organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like.

Pharmaceutically acceptable base addition salts include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. In some embodiments, the pharmaceutically acceptable baseaddition salts are the ammonium, potassium, sodium, calcium, andmagnesium salts. Base addition salts also include those derived frompharmaceutically acceptable organic non-toxic bases, including salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

Protecting group has the meaning conventionally associated with it inorganic synthesis, i.e. a group that selectively blocks one or morereactive sites in a multifunctional compound such that a chemicalreaction can be carried out selectively on another unprotected reactivesite and such that the group can readily be removed after the selectivereaction is complete. A variety of protecting groups are disclosed, forexample, in T. H. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, Third Edition, John Wiley & Sons, New York (1999), which isincorporated herein by reference in its entirety. For example, a hydroxyprotected form is where at least one of the hydroxyl groups present in acompound is protected with a hydroxy protecting group. Likewise, aminesand other reactive groups may similarly be protected.

Solvate refers to the compound formed by the interaction of a solventand a compound of Formula I or salt thereof, and as will be understoodby those skilled in the art, is the compound or salt with which asolvent is incorporated, for example, into the crystal structure.Suitable solvates of the compounds of the Formula I or a salt thereofare those formed with a pharmaceutically acceptable solvent, includinghydrates (i.e., wherein the solvent is water).

Many of the compounds described herein contain one or more asymmetriccenters (e.g. the carbon to which R₂ and R_(2′) are attached where R₂differs from R_(2′)) and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)— or (S)—. The presentinvention is meant to include all such possible isomers, includingracemic mixtures, optically pure forms and intermediate mixtures.Optically active (R)— and (S)-isomers may be prepared using chiralsynthons or chiral reagents, or resolved using conventional techniques.When the compounds described herein contain olefinic double bonds orother centers of geometric asymmetry, and unless specified otherwise, itis intended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms and rotational isomers are also intendedto be included.

When desired, the R— and S-isomers may be resolved by methods known tothose skilled in the art, for example by formation of diastereoisomericsalts or complexes which may be separated, for example, bycrystallization; via formation of diastereoisomeric derivatives whichmay be separated, for example, by crystallization, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example enzymatic oxidation orreduction, followed by separation of the modified and unmodifiedenantiomers; or gas-liquid or liquid chromatography in a chiralenvironment, for example on a chiral support, such as silica with abound chiral ligand or in the presence of a chiral solvent. It will beappreciated that where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step may be required to liberate the desired enantiomeric form.Alternatively, specific enantiomer may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer to the other by asymmetrictransformation.

The present invention is directed to a class of novel compounds, thatcan be described as isoquinolone derivatives, that are inhibitors of oneor more mitotic kinesins. In some embodiments, the compounds describedherein inhibit the mitotic kinesin, KSP, such as human KSP. In someembodiments, the compounds inhibit the mitotic kinesin, KSP, as well asmodulating one or more of the human mitotic kinesins selected from thegroup consisting of HSET (see, U.S. Pat. No. 6,361,993, which isincorporated herein by reference); MCAK (see, U.S. Pat. No. 6,331,424,which is incorporated herein by reference); CENP-E (see, PCT PublicationNo. WO 99/13061, which is incorporated herein by reference); Kif4 (see,U.S. Pat. No. 6,440,684, which is incorporated herein by reference);MKLP1 (see, U.S. Pat. No. 6,448,025, which is incorporated herein byreference); Kif15 (see, U.S. Pat. No. 6,355,466, which is incorporatedherein by reference); Kid (see, U.S. Pat. No. 6,387,644, which isincorporated herein by reference); Mpp1, CMKrp, KinI-3 (see, U.S. Pat.No. 6,461,855, which is incorporated herein by reference); Kip3a (see,PCT Publication No. WO 01/96593, which is incorporated herein byreference); Kip3d (see, U.S. Pat. No. 6,492,151, which is incorporatedherein by reference); and RabK6.

The methods of inhibiting a mitotic kinesin comprise contacting aninhibitor of the invention with a kinesin, such as a human kinesin, forexample, human KSP or fragments and variants thereof. The inhibition canbe of the ATP hydrolysis activity of the KSP kinesin and/or the mitoticspindle formation activity, such that the mitotic spindles aredisrupted. Meiotic spindles may also be disrupted.

The present invention provides inhibitors of mitotic kinesins, such asKSP, for example, human KSP, for the treatment of disorders associatedwith cell proliferation. The compounds described herein can differ intheir selectivity and are used to treat diseases of cellularproliferation, including, but not limited to cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders, fungal disorders andinflammation.

Accordingly, the present invention relates to methods employing at leastone chemical entity chosen from compounds of Formula I:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein,

-   -   W, X, Y, and Z are independently —N═, N, —C═, CR₁₈, O or S and Z        is optionally absent, provided that:        -   the ring comprising W, X, Y, and optionally Z is            heterocyclic or heteroaromatic;        -   no more than two of W, X, Y, and Z is —N═, and        -   W, X, or Y can be O or S only when Z is absent;    -   the dashed lines in the structure depict optional double bonds;    -   T and T′ are independently a covalent bond or optionally        substituted lower alkylene;    -   R₁ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-, optionally substituted aralkyl-,        optionally substituted heteroaryl-, and optionally substituted        heteroaralkyl-;    -   R₂ and R_(2′) are independently chosen from hydrogen, optionally        substituted alkyl-, optionally substituted aryl-, optionally        substituted aralkyl-, optionally substituted heteroaryl-, and        optionally substituted heteroaralkyl-; or R₂ and R_(2′) taken        together form an optionally substituted 3- to 7-membered ring;    -   R₁₂ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-optionally substituted aralkyl-,        optionally substituted heteroaryl-, optionally substituted        heteroaralkyl-, —C(O)—R₃, and —S(O)₂—R_(3a);    -   R₃ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-, optionally substituted aralkyl-,        optionally substituted heteroaryl-, optionally substituted        heteroaralkyl-, R₁₅O— and R₁₇—NH—;    -   R_(3a) is chosen from optionally substituted alkyl-, optionally        substituted aryl-, optionally substituted aralkyl-, optionally        substituted heteroaryl-, optionally substituted heteroaralkyl-,        and R₁₇—NH—;    -   R₄ is chosen from hydrogen, optionally substituted alkyl-,        optionally substituted aryl-, optionally substituted aralkyl-,        optionally substituted heteroaralkyl-, and optionally        substituted heterocyclyl-;    -   or R₄ taken together with R₁₂, and the nitrogen to which they        are bound, form an optionally substituted 5- to 12-membered        nitrogen-containing heterocycle, which optionally incorporates        one or two additional heteroatoms, selected from N, O, and S in        the heterocycle ring;    -   or R₄ taken together with R₂ form an optionally substituted 5-        to 12-membered nitrogen-containing heterocycle, which optionally        incorporates one or two additional heteroatoms, selected from N,        O, and S in the heterocycle ring;    -   R₅, R₆, R₇, R₈, and R₁₈ are independently chosen from hydrogen,        acyl, optionally substituted alkyl-, optionally substituted        alkoxy, halogen, hydroxyl, nitro, cyano, optionally substituted        amino, alkylsulfonyl-, alkylsulfonamido-, alkylthio-,        carboxyalkyl-, carboxamido-, aminocarbonyl-, optionally        substituted aryl and optionally substituted heteroaryl-,        provided that R₅, R₆, R₇ and R₈ is absent where W, X, Y, or Z,        respectively, is —N═, O, S or absent;    -   R₁₅ is chosen from optionally substituted alkyl-, optionally        substituted aryl-, optionally substituted aralkyl-, optionally        substituted heteroaryl-, and optionally substituted        heteroaralkyl-; and    -   R₁₇ is hydrogen, optionally substituted alkyl-, optionally        substituted aryl-, optionally substituted aralkyl-, optionally        substituted heteroaryl-, or optionally substituted        heteroaralkyl.

In some embodiments, the methods employ at least one chemical entitychosen from compounds of Formula II:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein R₁, R₂, R_(2′), R₄ through R₈, R₁₂, T, and T′ are as definedabove and provided that T and T′ are not both absent.

In some embodiments, the methods employ at least one chemical entitychosen from compounds of Formula III:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein

-   -   R₁, R₃, R_(3a), R₁₅, R₁₇ and R₅ through R₈ are as defined above;        and    -   either R₂ and R_(2′) are independently chosen from hydrogen,        optionally substituted alkyl-, optionally substituted aryl-,        optionally substituted aralkyl-, optionally substituted        heteroaryl-, and optionally substituted heteroaralkyl- or R₂ and        R_(2′) taken together form an optionally substituted 3- to        7-membered ring; and R₁₂ taken together with R₄, and the        nitrogen to which they are bound, form an optionally substituted        5- to 12-membered nitrogen-containing heterocycle, which        optionally incorporates one or two additional heteroatoms,        selected from N, O, and S in the heterocycle ring, provided that        such 5-membered nitrogen-containing heterocycle is not an        optionally substituted imidazolyl or imidazolinyl ring;    -   or R₂ taken together with R₄ form an optionally substituted 5-        to 12-membered nitrogen-containing heterocycle, which optionally        incorporates one or two additional heteroatoms, selected from N,        O, and S in the heterocycle ring; R_(2′) is hydrogen, optionally        substituted alkyl-, optionally substituted aryl-, optionally        substituted aralkyl-, optionally substituted heteroaryl-, or        optionally substituted heteroaralkyl; and R₁₂ is chosen from        hydrogen, optionally substituted alkyl-, optionally substituted        aryl-, optionally substituted aralkyl-, optionally substituted        heteroaryl-, optionally substituted heteroaralkyl-, —C(O)—R₃,        and —S(O)₂—R_(3a).

In certain embodiments, the stereogenic center to which R₂ and R_(2′)are attached is of the R configuration.

Compounds of Formula I, II, and III can be prepared by following theprocedures described with reference to the Reaction Schemes below.

Unless specified otherwise, the terms “solvent”, “inert organic solvent”or “inert solvent” mean a solvent inert under the conditions of thereaction being described in conjunction therewith [including, forexample, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”),dimethylformamide (“DMF”), chloroform, methylene chloride (ordichloromethane), diethyl ether, methanol, pyridine and the like].Unless specified to the contrary, the solvents used in the reactions ofthe present invention are inert organic solvents.

In general, esters of carboxylic acids may be prepared by conventionalesterification procedures, for example alkyl esters may be prepared bytreating the required carboxylic acid with the appropriate alkanol,generally under acidic conditions. Likewise, amides may be preparedusing conventional amidation procedures, for example amides may beprepared by treating an activated carboxylic acid with the appropriateamine. Alternatively, a lower-alkyl ester such as a methyl ester of theacid may be treated with an amine to provide the required amide,optionally in presence of trimethylalluminium following the proceduredescribed in Tetrahedron Lett. 48, 4171-4173, (1977). Carboxyl groupsmay be protected as alkyl esters, for example methyl esters, whichesters may be prepared and removed using conventional procedures, oneconvenient method for converting carbomethoxy to carboxyl is to useaqueous lithium hydroxide.

The salts and solvates of the compounds mentioned herein may as requiredbe produced by methods conventional in the art. For example, if aninventive compound is an acid, a desired base addition salt can beprepared by treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary, or tertiary); an alkalimetal or alkaline earth metal hydroxide; or the like. Illustrativeexamples of suitable salts include organic salts derived from aminoacids such as glycine and arginine; ammonia; primary, secondary, andtertiary amines; such as ethylenediamine, and cyclic amines, such ascyclohexylamine, piperidine, morpholine, and piperazine; as well asinorganic salts derived from sodium, calcium, potassium, magnesium,manganese, iron, copper, zinc, aluminum, and lithium.

If a compound is a base, a desired acid addition salt may be prepared byany suitable method known in the art, including treatment of the freebase with an inorganic acid, such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like, or withan organic acid, such as acetic acid, maleic acid, succinic acid,mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid,glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acidor galacturonic acid, alpha-hydroxy acid, such as citric acid ortartaric acid, amino acid, such as aspartic acid or glutamic acid,aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid,such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonicacid, or the like.

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

The compounds of Formula I, II, and III can be prepared by following theprocedures described in U.S. patent application Ser. No. 10/462,002 andcorresponding PCT Application No. PCT/US03/18778; U.S. patentapplication Ser. No. 10/366,828 and corresponding PCT Application No.US03/04713; PCT Publication Nos. WO 01/30768, WO 01/98278, PCT WO03/39460, WO 03/49678, WO 03/50122, WO 03/49527, WO 0349679, and WO03/50064, each of which is incorporated herein by reference for allpurposes, and with reference to the Reaction Schemes below.

The optionally substituted benzoic acids of Formula 101 and the otherreactants are commercially available, e.g., from Aldrich ChemicalCompany, Milwaukee, Wis. or may be readily prepared by those skilled inthe art using commonly employed synthetic methodology.

Preparation of Compounds of Fromula 103

Referring to Reaction Scheme 1, Step 1, an optionally substitutedcarboxylic acid of Formula 101 is protected with a suitable protectinggroup. For example, the following procedure is used to prepare esters ofthe carboxylic acid. A solution of a compound of Formula 101 and thionylchloride in a polar aprotic solvent (such as DMF) is warmed gently untilthe mixture becomes homogeneous. The solution is concentrated. A loweralkanol, such as methanol, is then added. The corresponding, optionallysubstituted compound of Formula 103 is isolated and purified.

Preparation of Compounds of Formula 105

Referring to Reaction Scheme 1, Step 2, a compound of Formula 103 iscoupled to a compound of Formula 104. A variety of reaction conditionscan be used to effect this coupling, e.g., Sonogashira couplingconditions using a palladium catalyst such as(diphenylphosphineferrocenyl)dichloropalladium or tris(dibenzylidenacetone)-dipalladium; a base such as cesium carbonate ortriethylamine; and ligands such as triphenylarsine ortriphenylphosphine; Stille conditions using a palladium catalyst such astris (dibenzylidinacetone)-dipalladium; lithium chloride; andtriphenylarsine; or Suzuki coupling conditions wherein the compound ofFormula 104 is treated with a borane such as catechol borane or9-borabicyclo[3.3.1]nonane and the two fragments are coupled using apalladium catalyst such as those described above, a base such as cesiumcarbonate, and triphenylarsine.

In some embodiments, a mixture of a compound of Formula 103; a slightexcess (such as about 1.1 equivalents) of an acetylenic compound ofFormula 104; a palladium catalyst such asdichlorobis(triphenylphosphine)palladium(II), for example, about 0.025equivalents of the catalyst; and cuprous iodide in a base such astriethylamine is heated at about 50° C. The corresponding, optionallysubstituted compound of Formula 105 is isolated and purified.

Preparation of Compounds of Formula 107

Referring to Reaction Scheme 1, Step 3, the protecting group is thenremoved from the carboxylic acid. When the protecting group is a loweralkyl ester, it can be removed by treatment with aqueous alcoholic baseat an elevated temperature. For example, methyl esters can be convertedto the corresponding carboxylic acid by treatment of the ester withpotassium hydroxide in a 1:1:1 solution of methanol, THF, and alcohol atabout 50° C. for 30 minutes. The corresponding, optionally substitutedcompound of Formula 107 is isolated and used without furtherpurification.

Preparation of Compounds of Formula 109

Referring to Reaction Scheme 1, Step 4,bis(acetonitrile)dichloropalladium (about 0.050 equivalents) is added toa solution of a compound of Formula 107 and a base, such astriethylamine, in a nonpolar, aprotic solvent such as THF. The solutionis maintained at an elevated temperature, such as about 50° C. Thecorresponding, optionally substituted isochromen-1-one of Formula 109 isisolated and purified.

Preparation of Compounds of Formula 111

Referring to Reaction Scheme 1, Step 5, a solution of isochromen-1-oneof Formula 109 and an excess (such as about 3 equivalents) of a primaryamine of formula R₁NH₂ in a nonpolar solvent such as toluene is heatedat reflux, such as about 140° C. The resulting amide is isolated anddissolved in a polar, protic solvent such as methanol. Aqueous acid,such as about 5% aqueous hydrochloric acid is added to the solutionwhich is then heated to about 50° C. Additional aqueous alcholic acidmay be added if required to complete the reaction. The corresponding,optionally substituted 2H-isoquinolin-1-one of Formula 111 is isolatedand purified.

Preparation of Compounds of Formula 113

Referring to Reaction Scheme 1, Step 6, the protecting group is removedfrom the primary amine. In some embodiments, the protecting group is Bocand its removal can be accomplished through treatment with aqueous TFAat room temperature. The product, a compound of Formula 113 is isolatedand can be used without further purification.

In certain embodiments, a particular stereoconfiguration for the R₂substituent, such as the (R) isomer, can be obtained. An amine ofFormula 113 is dissolved in an inert organic solvent (such as IPA) andwarmed to 60° C. In a separate vessel, a resolving agent (such asdibenzoyl-D-tartaric acid) is dissolved, such as in the same warmsolvent, and then quickly added (with agitation) to the warm aminesolution. The reaction mixture is left to crystallize by cooling to roomtemperature over 16 hours under continuing agitation. The desiredisomer, e.g., the (R) isomer is isolated and purified in the usualmanner.

For the sake of brevity in the remaining description of the synthesis ofcompounds of Formula I, II, or III, it should be understood that eithersingle isomer or a mixture of isomers may be employed to give thecorresponding product.

Preparation of Formula 203

Referring to Reaction Scheme 2, Step 1, to a solution of a compound ofFormula 113 is added successively a slight excess (such as about 1.2equivalents) of an aldehyde comprising R_(4′) (i.e., a compound havingthe formula R_(4′)CHO where R_(4′)CH₂— is equivalent to R₄ and R₄ is asdescribed above or is a protected precursor to such a substituent, e.g.,(3-oxo-propyl)-carbamic acid tert-butyl ester) and a reducing agent suchas sodium triacetoxyborohydride. The resulting mixture is stirred forseveral hours. The product, a compound of Formula 203 is isolated andpurified.

Preparation of Formula 205

Referring to Reaction Scheme 2, Step 2, to a solution of a compound ofFormula 203 and an amine base such as diisopropylethylamine in anonpolar, aprotic solvent such as dichloromethane is added an R₃ acylchloride (such as Cl—C(O)—R₃ where R₃ is as described above). Theresulting solution is stirred under nitrogen at room temperature forseveral hours. The product, a compound of Formula 205 is isolated andpurified.

Optionally, any protecting groups on compounds of Formula 205 are thenremoved. For example, if R₄ comprises a protected amine wherein theprotecting group is a Boc group, the Boc group can be removed bytreatment of the compound of Formula 205 with an acid such astrifluoroacetic acid in a nonpolar, aprotic solvent such asdichloromethane, while maintaining the reaction at about roomtemperature. The reaction is monitored e.g., by TLC. Upon completion,the product is isolated and purified.

Referring to Reaction Scheme 3, to a solution of a compound of Formula203 and an amine base such as diisopropylethylamine in a nonpolar,aprotic solvent such as dichloromethane is added a compound having theformula Cl—S(O)₂—R_(3a) or O—(S(O)₂—R_(3a))₂ where R_(3a) is asdescribed above. The resulting solution is stirred under nitrogen atroom temperature for several hours. The product, a compound of Formula303 is isolated and purified.

Referring to Reaction Scheme 4, to a solution of a compound of Formula203 and an amine base such as diisopropylethylamine in a nonpolar,aprotic solvent such as dichloromethane is added a compound having theformula X—R₁₂ where R₁₂ is as described above and X is a leaving group.The resulting solution is stirred under nitrogen at room temperature orwith heat for several hours. The product, a compound of Formula 403 isisolated and purified.

Preparation of Formula 503

Referring to Reaction Scheme 5, Step 1, to an optionally substitutedcompound of Formula 113 dissolved in a polar, aprotic solvent (such asDMF) in the presence of a base (such as potassium carbonate) is addedone equivalent of an optionally substituted suitably protected aldehydewherein such aldehyde further comprises a leaving group, such as ahalide. The solution is heated at reflux, monitoring completion of thereaction (e.g., by TLC). The reaction mixture is cooled and thecorresponding, optionally substituted compound of Formula 503 isisolated and purified.

Preparation of Formula 505

Referring to Reaction Scheme 5, Step 2, to an optionally substitutedcompound of Formula 503 in an inert solvent (such as dichloromethane) inthe presence of about 1.5 molar equivalents of an amine base (such astriethylamine) is added about 1.5 molar equivalents of an R₉ acidchloride, such as, Cl—C(O)—R₉, where R₉ is as described herein. Thereaction takes place, with stirring, at room temperature over a periodof 4 to 24 hours. Completion is monitored, e.g., by TLC. Thecorresponding compound of Formula 505 is isolated and purified.

Preparation of Formula 507

Referring to Reaction Scheme 5, Step 3, a solution of a compound ofFormula 505 and an excess of ammonium acetate in acetic acid is heatedat reflux for 1-4 hours. Completion is monitored, e.g., by TLC. Thecorresponding compound of Formula 507 is isolated and purified.

Preparation of Formula 603

Referring to Reaction Scheme 6, Step 1, a suspension of a compound ofFormula 113, an alpha-haloketone reagent of the Formula R_(13′)(CO)CH₂Xwherein X is a leaving group (such as a halide) and R_(13′) is asdescribed herein, and about an equivalent of a base, such as potassiumcarbonate in a polar, aprotic solvent such as DMF is stirred at roomtemperature. The reaction is diluted with water and the resulting solid,a compound of Formula 603, is used in the subsequent step withoutfurther purification.

Preparation of Formula 605

Referring to Reaction Scheme 6, Step 2, a solution of the compound ofFormula 603, about an equivalent of an amine base, such as triethylamineand about an equivalent of an acid chloride (such as a compound ofFormula R₉—COCl) in an organic solvent such as methylene chloride isstirred at room temperature for several hours. Completion is monitored,e.g., by TLC. The corresponding compound of Formula 605 is isolated andpurified.

Preparation of Formula 607

Referring to Reaction Scheme 6, Step 3, a solution of a compound ofFormula 605 and an excess of ammonium acetate in acetic acid is heatedat reflux using a Dean-Stark trap and condenser. Completion ismonitored, e.g., by TLC. The corresponding compound of Formula 607 isisolated and purified.

Optionally, in the event that group R_(13′) comprises a functionalitybearing a protecting group, the protecting group is removed. Thus, ifR_(13′) further comprises an amine bearing a Pht group, the protectinggroup is removed by treatment of a solution of a compound of Formula 607with an excess of anhydrous hydrazine in a polar, protic solvent such asethanol. The solution is refluxed and then cooled to about 50° C. andany precipitate is filtered off. The filtrate is concentrated in vacuoand purified to yield the corresponding free amine.

Preparation of Formula 703

Referring to Reaction Scheme 7, Step 1, reductive amination of amines ofFormula 113 (prepared as described in WO 0130768) with an optionallysubstituted, aldehyde-containing carbamic acid ester (Seki et. al. Chem.Pharm. Bull. 1996, 44, 2061) gives urethane intermediates. Removal ofthe Boc protecting group furnishes an amine of Formula 705.

More specifically, to a solution of a compound of Formula 113 and anequivalent of a suitably protected aldehyde (Seki et. al. Chem. Pharm.Bull. 1996, 44, 2061) in dichloromethane is added a slight excess of areducing agent, such as sodium triacetoxyborohydride. The resultantcloudy mixture is maintained at ambient temperature. Completion ismonitored, e.g., by TLC. The corresponding compound of Formula 703 isisolated and used in the subsequent step without purification.

Preparation of Formula 705

Referring to Reaction Scheme 7, Step 2, the amine protecting group, PG,is then removed. For example, when PG is Boc, to a solution of acompound of Formula 703 in a nonpolar, aprotic solvent such asdichloromethane is added a strong acid such as trifluoroacetic acid. Theresultant solution is maintained at ambient temperature overnight andconcentrated under reduced pressure. The residue is isolated to give acompound of Formula 705 which was used in the subsequent step withoutpurification.

Preparation of Formula 707

Referring to Reaction Scheme 7, Step 3, to a solution of a compound ofFormula 705 in a nonpolar, aprotic solvent such as dichloromethane isadded an excess, such as about two equivalents, of an amine base such astriethylamine, followed by about an equivalent or slight excess of anacid chloride of the formula R₁₄COCl. The resultant solution is stirredat ambient temperature for about 3 hours. Completion is monitored, e.g.,by TLC. The corresponding compound of Formula 707 is isolated andpurified.

Preparation of Formula 709

Referring to Reaction Scheme 7, Step 4, a solution of a compound ofFormula 707 in an excess of phosphorus oxychloride is heated at reflux.After 8 hours, the reaction mixture is allowed to cool to ambienttemperature and concentrated under reduced pressure. The correspondingcompound of Formula 709 is isolated and purified.

Preparation of Formula 709

As an alternative to Steps 3 and 4 of Reaction Scheme 7, acylation ofprimary amines of Formula 705, followed by acetic acid mediatedcyclization, can proceed without isolation of the intermediate amides toprovide the target compound of Formula 709. This route is shown inReaction Scheme 8.

More specifically, to a solution of a compound of Formula 705 in anonpolar, aprotic solvent such as dichloromethane is added an excess,such as about two equivalents of an amine base, such as triethylamine,followed by about an equivalent of an acid chloride of formula R₁₄COCl.The resultant solution is stirred at ambient temperature for 2 hours,then evaporated under reduced pressure. The resultant solid is treatedwith glacial acetic acid, then the resultant suspension is heated atreflux for about 48 hours. The reaction is cooled to ambient temperaturethen evaporated under reduced pressure. The corresponding compound ofFormula 709 is isolated and purified.

Referring to Reaction Scheme 9, a compound of Formula 203 is reactedwith a slight excess of a compound of the formula R₁₅O(CO)Cl in thepresence of a base such as triethylamine in a nonpolar, aprotic solventsuch as dichloromethane. The product, a compound of Formula 903 isisolated and purified.

Referring to Reaction Scheme 10, a compound of Formula 203 is treatedwith a slight excess of an isocyanate R₁₇—N═C═O in the presence of abase, such as triethylamine, in a nonpolar, aprotic solvent, such asdichloromethane. The product, a compound of Formula 1003, is isolatedand purified.

Preparation of Compounds of Formula 1103

Referring to Reaction Scheme 11, to a solution of a compound of Formula1101 in a nonpolar, aprotic solvent such as DMF are added a base such astriethylamine and an excess (such as about 1.5 equivalents) of imidazolefollowed by about an equivalent of tetrabutylammonium iodide. Theresultant solution is heated to about 90° C., stirred for about 18 h andallowed to cool to room temperature. The product, a compound of Formula1103, is isolated and purified.

Preparation of Compounds of Formula 1203

Referring to Reaction Scheme 12, Step 1, to a solution of a compound ofFormula 113 and an excess of an optionally substituted,aldehyde-containing carbamic acid ester in a nonpolar, aprotic solventsuch as CH₂Cl₂ is added sodium triacetoxyborohydride. The mixture isstirred overnight. The product, a compound of Formula 1203, is isolatedand purified.

Preparation of Compounds of Formula 1207

Referring to Reaction Scheme 12, Step 2, to a solution of a compound ofFormula 1203 in a nonpolar, aprotic solvent such as toluene is added abase such as triethylamine followed by dropwise addition of an excess ofan acid chloride of the formula R₁₄—COCl. The reaction mixture is heatedto about 80° C. for about 18 h, then at reflux for about 4 h. Theproduct, a compound of Formula 1207, is isolated and purified.

Alternative Preparation of Compounds of Formula 1207

Referring to Reaction Scheme 12, Step 3, in some embodiments, protectinggroup, PG, must be removed separately prior to cyclization to form theimidazolinyl compound. For example, when PG is Boc, a solution of acompound of Formula 1205 in a solvent such as CH₂Cl₂/TFA (such as about4:1 CH₂Cl₂/TFA) is stirred at room temperature. The reaction mixture isconcentrated under reduced pressure and the residue is diluted with anonpolar, aprotic solvent such as CH₂Cl₂ and washed with aqueous base.The aqueous layer is extracted with a nonpolar, aprotic solvent such asCH₂Cl₂ and the combined extracts are dried, filtered and concentratedunder reduced pressure. The residue is diluted with a nonpolar, aproticsolvent such as THF and aqueous base (such as saturated aqueous NaHCO₃).The mixture is stirred at room temperature for 10 days. The product, acompound of Formula 1207, is isolated and purified.

Referring to Reaction Scheme 13, reductive amination of the primaryamino group in compounds of Formula 113 with (2-oxo-ethyl)-carbamic acidtert-butyl ester gives the corresponding secondary amine. Acylation withacryloyl chloride followed by deprotection of the tertiary amide andbase mediated cyclisation gives the desired diazepanones. If desired,further functionalization of the basic amine can be accomplished underconditions well known to those skilled in the art.

Referring to Reaction Scheme 14, reductive amination of the primaryamino group in compounds of Formula 113 with (2-oxo-ethyl)-carbamic acidtert-butyl ester gives the corresponding secondary amine. Acylation withchloropivaloyl chloride followed by deprotection of the tertiary amideand base mediated cyclisation gives the desired diazepanones. Ifdesired, further functionalization of the basic amine can beaccomplished under conditions well known to those skilled in the art.

Referring to Reaction Scheme 15, a compound of Formula 1501, one-halfmolar equivalent of an optionally substituted piperazine or diazepam (asshown above, where R₃₂ is as described herein) and an excess ofpotassium carbonate are combined in an organic solvent (e.g.,acetonitrile). The reaction takes place under a nitrogen atmosphere atelevated temperature (e.g., 100° C.) over a period of 8 hours, followedat a somewhat lower temperature (e.g., 60° C.) for a period of 5 days.The product, a compound of Formula 1503, is isolated and purified.

Optionally, in the event that R₃₂ is an amine protecting group, such asBoc, it may be removed by for example treatment with a 95/5 mixture ofTFA/water followed by stirring at room temperature for 1 hour. Theproduct, a compound of Formula 1803 wherein R₃₂ is hydrogen, can beisolated and purified. If desired, further functionalization of thebasic amine could be accomplished under conditions well known to thoseskilled in the art.

The synthesis of compounds of Formula I, II, or III wherein R₄ takentogether with R₂ form an optionally substituted 5- to 12-memberednitrogen-containing heterocycle can be accomplished according to thegeneral procedure shown in Scheme 16 and as described further inReaction Scheme 1 above.

A compound of Formula 203:

(where R₄ is optionally protected) is contacted with a slight molarexcess of an R₃ chloride [such as, Cl—C(O)—R₃, Cl—S(O)₂—R_(3a), Cl—R₃,Cl—C(O)—O—R₁₅ and Cl—S(O)₂—NH—R_(3a)] or an isocyanate (such asO═C═N—R₁₇) or an anhydride (such as O[C(O)R₁₅]₂ or O[S(O)₂R_(3a)]₂) togive the corresponding optionally protected compound of Formula I, II,or III.

A compound of Formula 505, 605, 705, or 707 is optionally cyclized byacid-mediated cyclization.

A racemic mixture of isomers of a compound of Formula I, II, or III isoptionally placed on a chromatography column and separated into (R)— and(S)-enantiomers.

A compound of Formula I, II, or III is optionally contacted with apharmaceutically acceptable acid or base to form the corresponding acidor base addition salt.

A pharmaceutically acceptable acid addition salt of a compound ofFormula I, II, or III is optionally contacted with a base to form thecorresponding free base of Formula I, II, or III.

A pharmaceutically acceptable base addition salt of a compound ofFormula I, II, or III is optionally contacted with an acid to form thecorresponding free acid of Formula I, II, or III.

T and T′

When considering the compounds of Formula I or II, in some embodiments,T is optionally substituted alkylene or is absent (i.e., is a covalentbond); and T′ is optionally substituted alkylene or is absent (i.e., isa covalent bond). In some embodiments, one of T and T′ is absent and theother is optionally substituted alkylene (such as optionally substitutedmethylene). In some embodiments, both are absent. In some embodimentsboth are optionally substituted alkylene.

R₁

When considering the compounds of Formula I, II, or III, in someembodiments R₁ is selected from hydrogen, optionally substituted C₁-C₈alkyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted aryl-C₁-C₄-alkyl-, and optionally substitutedheteroaryl-C₁-C₄-alkyl- (such as optionally substituted aryl andoptionally substituted aryl-C₁-C₄-alkyl-). In some embodiments, R₁ isselected from hydrogen, optionally substituted C₁-C₄ alkyl, optionallysubstituted phenyl-C₁-C₄-alkyl-, optionally substitutedheteroaryl-C₁-C₄-alkyl-, optionally substituted naphthalenylmethyl,optionally substituted phenyl, and naphthyl. In some embodiments, R₁ isoptionally substituted phenyl-C₁-C₄-alkyl-, optionally substitutedheteroaryl-C₁-C₄-alkyl-, optionally substituted naphthalenylmethyl,optionally substituted phenyl, or naphthyl (such as optionallysubstituted phenyl-C₁-C₄-alkyl-, optionally substitutedheteroaryl-C₁-C₄-alkyl-).

In some embodiments, R₁ is naphthyl, phenyl, bromophenyl, chlorophenyl,methoxyphenyl, ethoxyphenyl, tolyl, dimethylphenyl, chorofluorophenyl,methylchlorophenyl, ethylphenyl, phenethyl, benzyl, halobenzyl (such aschlorobenzyl or bromobenzyl), methylbenzyl, methoxybenzyl, cyanobenzyl,hydroxybenzyl, dichlorobenzyl, dimethoxybenzyl, or naphthalenylmethyl.In some embodiments, R₁ is benzyl, halobenzyl, methylbenzyl,hydroxybenzyl, cyanobenzyl, methoxybenzyl, or naphthalenylmethyl. Insome embodiments, R₁ is benzyl.

R₂ and R_(2′)

When considering the compounds of Formula I, II, or III and as will beappreciated by those skilled in the art, the compounds described hereinpossess a potentially chiral center at the carbon to which R₂ and R_(2′)are attached. The R₂ and R_(2′) groups may be the same or different; ifdifferent, the compound is chiral (i.e., has a stereogenic center). WhenR₂ and R_(2′) are different, in some embodiments R_(2′) is hydrogen andR₂ is other than hydrogen. The invention contemplates the use of pureenantiomers and mixtures of enantiomers, including racemic mixtures. Theterm “substantially pure” means having at least about 95% chemicalpurity with no single impurity greater than about 1%. The term“substantially optically pure” or “enantiomerically pure” means havingat least about 97.5% enantiomeric excess. In some embodiments, thestereogenic center to which R₂ and R_(2′) are attached is of the Rconfiguration.

When considering the compounds of Formula I, in some embodiments R₂ andR_(2′) are independently chosen from hydrogen, optionally substitutedalkyl-, optionally substituted alkoxy, optionally substituted aryl-,optionally substituted aralkyl-, optionally substituted heteroaryl-, andoptionally substituted heteroaralkyl-; or R₂ and R_(2′) taken togetherform an optionally substituted 3- to 7-membered ring.

In some embodiments, R₂ is optionally substituted C₁-C₄ alkyl-, and R₂is hydrogen or optionally substituted C₁-C₄ alkyl-. In some embodiments,R_(2′) is hydrogen and R₂ is optionally substituted C₁-C₄ alkyl-. Insome embodiments, R₂ is chosen from methyl-, ethyl-, propyl (such asc-propyl or i-propyl), butyl (such as t-butyl), methylthioethyl-,methylthiomethyl-, aminobutyl-, (CBZ)aminobutyl-, cyclohexylmethyl-,benzyloxymethyl-, methylsulfanylethyl-, methylsulfanylmethyl-, andhydroxymethyl-, and R_(2′) is hydrogen. In some embodiments, R_(2′) ishydrogen and R₂ is ethyl or propyl (such as c-propyl or i-propyl). Insome embodiments, R₂ is i-propyl. In some embodiments, the stereogeniccenter to which R₂ and R_(2′) is attached is of the R configuration.

In some embodiments, if either R₂ or R_(2′) is hydrogen, then the otheris not hydrogen. In some embodiments, both R₂ and R_(2′) are hydrogen.

R₂ Taken Together with R₄

When considering the compounds of Formula I, II, or III, in someembodiments, R₂ and R₄ taken together form a 5- to 12-membered ringwhich optionally incorporates one or two additional heteroatoms,selected from N, O, and S in the heterocycle ring and may optionally besubstituted with one or more of the following groups: alkyl, aryl,aralkyl, heteroaryl, substituted alkyl, substituted aryl, substitutedaralkyl, and substituted heteroaryl, hydroxyl, alkoxy, cyano, optionallysubstituted amino, oxo, or carbamyl.

In some embodiments of compounds of Formula I or II, R₂ and R₄ takentogether form an optionally substituted ring of the formula:

wherein R₄₁ and R_(41′) are independently chosen from hydrogen, alkyl,aryl, aralkyl, heteroaryl, substituted alkyl, substituted aryl,substituted aralkyl, and substituted heteroaryl; m is 0, 1, 2, or 3; andT, T′, R₁₂, and R_(2′) are as defined above. In some embodiments, R₄₁ ishydrogen. In some embodiments, both R₄₁ and R_(41′) are hydrogen. Insome embodiments, R₁₂ is optionally substituted aralkyl (such as benzyl)or optionally substituted acyl (i.e., R₁₂ is —(CO)R₃ where R₃ is asdefined above, for example, R₃ is optionally substituted phenyl). See,e.g., PCT/US03/030788, which is incorporated herein by reference for allpurposes.

In some embodiments of compounds of Formula III, R₂ and R₄ takentogether form an optionally substituted ring of the formula:

wherein R₄₁ and R_(41′) are independently chosen from hydrogen, alkyl,aryl, aralkyl, heteroaryl, substituted alkyl, substituted aryl,substituted aralkyl, and substituted heteroaryl; m is 1, 2, or 3; andR₁₂ and R_(2′) are as defined above. In some embodiments, R₄₁ ishydrogen. In some embodiments, both R₄₁ and R_(41′) are hydrogen. Insome embodiments, R₁₂ is optionally substituted aralkyl (such as benzyl)or optionally substituted acyl (i.e., R₁₂ is —(CO)R₃ where R₃ is asdefined above, for example, R₃ is optionally substituted phenyl). See,e.g., U.S. Ser. No. PCT/US03/030788, which is incorporated herein byreference for all purposes.

In some embodiments of compounds of Formula I or II, R₂ and R₄ takentogether form an optionally substituted ring of the formula:

wherein R₁₂, R_(2′), T, and T′ are as defined above; R₅₁ and R_(51′) areindependently chosen from hydrogen, alkyl, aryl, aralkyl, heteroaryl,substituted alkyl, substituted aryl, substituted aralkyl and substitutedheteroaryl; U is a covalent bond, CR′R″ or NR′″; R′ and R″ areindependently chosen from hydrogen, hydroxy, amino, optionallysubstituted aryl, optionally substituted alkylamino, optionallysubstituted alkyl and optionally substituted alkoxy; and R′″ is chosenfrom hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroaryl,and optionally substituted heteroaralkyl.

In some embodiments, R₅₁ is hydrogen or optionally substituted loweralkyl. In some embodiments, R₅₁ is hydrogen. In some embodiments,R_(51′) is hydrogen or optionally substituted lower alkyl. In someembodiments, R_(51′) is hydrogen.

In some embodiments, R₁₂ is optionally substituted aryl or optionallysubstituted aralkyl. In some embodiments, R₁₂ is optionally substitutedphenyl, benzyl or methyl-benzyl (such as benzyl or methyl-benzyl).

In some embodiments, U is CR′R″ where R′ and/or R″ are hydrogen. In someembodiments, U is NR′″ where R′″ is hydrogen or optionally substitutedalkyl. In some embodiments, R′″ is hydrogen or optionally substitutedamino-lower alkyl. See, e.g., PCT/US03/23319, which is incorporatedherein by reference for all purposes.

In some embodiments of compounds of Formula III, R₂ and R₄ takentogether form an optionally substituted ring of the formula:

wherein R₁₂ and R_(2′) are as defined above; R₅₁ and R_(51′) areindependently chosen from hydrogen, alkyl, aryl, aralkyl, heteroaryl,substituted alkyl, substituted aryl, substituted aralkyl and substitutedheteroaryl; U is a covalent bond, CR′R″ or NR′″; R′ and R″ areindependently chosen from hydrogen, hydroxy, amino, optionallysubstituted aryl, optionally substituted alkylamino, optionallysubstituted alkyl and optionally substituted alkoxy; and R′″ is chosenfrom hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroaryl,and optionally substituted heteroaralkyl. In some embodiments, R₅₁ ishydrogen or optionally substituted lower alkyl. In some embodiments, R₅₁is hydrogen. In some embodiments, R_(51′) is hydrogen or optionallysubstituted lower alkyl. In some embodiments, R_(51′) is hydrogen. Insome embodiments, R₁₂ is optionally substituted aryl or optionallysubstituted aralkyl. In some embodiments, R₁₂ is optionally substitutedphenyl, benzyl or methyl-benzyl (such as benzyl or methyl-benzyl). Insome embodiments, U is CR′R″ where R′ and/or R″ are hydrogen. In someembodiments, U is NR′″ where R′″ is hydrogen or optionally substitutedalkyl. In some embodiments, R′″ is hydrogen or optionally substitutedamino-lower alkyl. See, e.g., PCT/US03/23319, which is incorporatedherein by reference for all purposes.R₅, R₆, R₇, R₈, and R₁₈

When considering the compounds of Formula I, II, and III, in someembodiments, R₅, R₆, R₇, R₈ and R₁₈ are independently chosen fromhydrogen, acyl, optionally substituted alkyl-, optionally substitutedalkoxy, halogen, hydroxyl, nitro, cyano, optionally substituted amino,alkylsulfonyl-, alkylsulfonamido-, alkylthio-, carboxyalkyl-,carboxamido-, aminocarbonyl-, optionally substituted aryl and optionallysubstituted heteroaryl-, provided that R₅, R₆, R₇ and R₈ is absent whereW, X, Y, or Z, respectively, is —N═, O, S or absent. In someembodiments, when present, R₅, R₆, R₇, R₈ and R₁₈ are independentlychosen from hydrogen, hydroxyl, halogen (such as chloro and fluoro),optionally substituted C₁-C₄ alkyl- (such as methyl-), C₁-C₄ alkoxy(such asmethoxy), cyano, amino, substituted amino, or carbamyl-. In someembodiments, R₅, R₆, R₇, R₈ and R₁₈ are methoxy, methyl,trifluoromethyl, cyano, hydrogen or halo. In some embodiments, R₅ ishydrogen or halo; R₆ is hydrogen, optionally substituted C₁-C₄ alkyl-(such as methyl-) or halo; R₇ is hydrogen, halo, optionally substitutedC₁-C₄ alkyl- (such as methyl- or trifluoromethyl-), C₁-C₄ alkoxy (suchas methoxy), cyano, substituted amino, or carbamyl-; and R₈ is hydrogen,C₁-C₄ alkyl- (such as methyl-), C₁-C₄ alkoxy (such as methoxy), hydroxy,or halo. In some embodiments, only one of R₅, R₆, R₇, and R₈ is nothydrogen, such as R₇. In some embodiments, R₇ and R₈ are not hydrogen.In some embodiments, R₁₈ is hydrogen.

R₄ Taken Together with R₁₂

When considering the compounds of Formula I, II, or III, in someembodiments, R₄ taken together with R₁₂, and the nitrogen to which theyare bound, form an optionally substituted 5- to 12-memberednitrogen-containing heterocycle, which optionally incorporates one ortwo additional heteroatoms, selected from N, O, and S in the heterocyclering and may optionally be substituted with one or more of the followinggroups: alkyl, aryl, aralkyl, heteroaryl, substituted alkyl, substitutedaryl, substituted aralkyl, and substituted heteroaryl, hydroxyl, alkoxy,cyano, optionally substituted amino, oxo, or carbamyl.

When considering the compounds of Formula I and in some embodiments ofcompounds of Formula II when T and T′ are not both absent, R₄ takentogether with R₁₂ and the nitrogen to which they are bound, forms anoptionally substituted imidazolyl ring of the formula:

wherein

-   -   R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl-,        optionally substituted aryl-, optionally substituted        aryl-C₁-C₄-alkyl-, optionally substituted        heteroaryl-C₁-C₄-alkyl-, optionally substituted        aryl-C₁-C₄-alkoxy, optionally substituted        heteroaryl-C₁-C₄-alkoxy, and optionally substituted heteroaryl-;        and    -   R₁₃ and R_(13′) are independently hydrogen, optionally        substituted C₁-C₈ alkyl-, optionally substituted aryl-, or        optionally substituted aryl-C₁-C₄-alkyl-. See, e.g.,        PCT/US03/14787, which is incorporated herein by reference.

In some embodiments, R₉ is phenyl substituted with C₁-C₄-alkyl-,C₁-C₄-alkoxy-, and/or halo; phenyl-; benzyl-; thienyl-; or thienyl-substituted with C₁-C₄-alkyl-, C₁-C₄-alkoxy-, and/or halo. In someembodiments, R₉ is phenyl substituted with one or more halo and/ormethyl.

In some embodiments, R₁₃ is hydrogen and R_(13′) is substituted C₁-C₄alkyl-. In some embodiments, R₁₃ is hydrogen and R_(13′) isaminomethyl-, aminoethyl-, aminopropyl-, acetylamino-methyl-,acetylaminoethyl-, benzyloxycarbonylamino-methyl- orbenzyloxycarbonylamino-ethyl-.

When considering the compounds of Formula I and in some embodiments ofcompounds of Formula II when T and T′ are not both absent, R₄ takentogether with R₁₂ forms an optionally substituted imidazolinyl ring ofthe formula:

-   -   wherein,    -   R₁₄ is chosen from hydrogen, optionally substituted C₁-C₈        alkyl-, optionally substituted aryl-, optionally substituted        aryl-C₁-C₄-alkyl-, optionally substituted heteroaryl-,        optionally substituted heteroaryl-C₁-C₄-alkyl-; and    -   R₁₀, R_(10′), R₁₁, and R_(11′) are independently chosen from        hydrogen, optionally substituted C₁-C₈ alkyl-, optionally        substituted aryl-, and optionally substituted aryl-C₁-C₄-alkyl-.

In some embodiments, R₁₄ is methylenedioxyphenyl-; phenyl-; phenylsubstituted with C₁-C₄ alkyl-, C₁-C₄ alkoxy-, and/or halo; benzyl-;thienyl substituted with C₁-C₄ alkyl; benzyl; thiophenyl-; orthiophenyl-substituted with C₁-C₄-alkyl-, C₁-C₄-alkoxy-, and/or halo. Insome embodiments, R₁₄ is methylenedioxyphenyl-; phenyl-; tolyl-;methoxyphenyl-; or halomethylphenyl-.

In some embodiments, R₁₀, R_(10′), R_(11′), and R₁₁ are independentlyhydrogen or optionally substituted C₁-C₄ alkyl-. In some embodiments,R_(11′) and R₁₁ are hydrogen.

When considering the compounds of Formula I, II, or III, in someembodiments, R₄ taken together with R₁₂ forms an optionally substituteddiazepinone ring of the formula:

wherein A and B are each independently chosen from C(R₂₀)(R₂₁), N(R₂₂),O or S, wherein R₂₀ and R₂₁ are each independently selected from H,optionally substituted alkyl optionally substituted aryl and optionallysubstituted heteroaryl; and R₂₂ is H, optionally substituted alkyl,optionally substituted aralkyl, optionally substituted heteroaralkyl,optionally substituted alkylcarbonyl, optionally substitutedarylcarbonyl, optionally substituted heteroarylcarbonyl, optionallysubstituted aralkylcarbonyl, optionally substitutedheteroaralkylcarbonyl, optionally substituted alkoxycarbonyl, optionallysubstituted aryloxycarbonyl, optionally substitutedheteroaryloxycarbonyl, optionally substituted aralkyloxycarbonyl,optionally substituted heteroaralkyloxycarbonyl. In some embodiments,the diazepinone ring is further substituted with one or more of thefollowing groups: optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroaryl,and optionally substituted heteroaralkyl.

In some embodiments of the compounds of Formula I, II, or III, one of Aor B is C(R₂₀)(R₂₁), wherein R₂₀ and R₂₁ are each independently selectedfrom H or C₁-C₄ alkyl, and the other of A or B is N(R₂₂), where R₂₂ isH, C₁-C₄ alkyl, optionally substituted aralkyl, optionally substitutedheteroaralkyl, C₁-C₆ alkylcarbonyl, optionally substituted arylcarbonyl,optionally substituted heteroarylcarbonyl, optionally substitutedaralkylcarbonyl, optionally substituted heteroaralkylcarbonyl, C₁-C₆alkoxycarbonyl, optionally substituted aryloxycarbonyl, optionallysubstituted heteroaryloxycarbonyl, optionally substitutedaralkyloxycarbonyl, optionally substituted heteroaralkyloxycarbonyl,where the optionally substituted aryl or heteroaryl groups or moietiesare unsubstituted or substituted with one or more substituents selectedfrom C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy,amino, C₁-C₄ alkylamino, di-C₁-C₄ alkylamino, carboxy, C₁-C₄alkylcarbonyloxy, C₁-C₄ alkoxycarbonyl, carboxamido, C₁-C₄alkylcarboxamido, aminocarbonyl, C₁-C₄ alkylaminocarbonyl, di-C₁-C₄alkylaminocarbonyl, cyano, C₁-C₄ alkylcarbonyl, halogen, hydroxyl,mercapto and nitro. In some embodiments, A is C(R₂₀)(R₂₁), wherein R₂₀and R₂₁ are each H or C₁-C₄ alkyl, and B is N(R₂₂), where R₂₂ is H,C₁-C₄ alkyl, aralkyl, heteroaralkyl, C₁-C₆ alkylcarbonyl, arylcarbonyl,heteroarylcarbonyl. In some embodiments of the compounds of Formula I, Ais CH₂, and B is N(R₂₂), where R₂₂ is H, methyl, benzyl or acetyl(—C(O)methyl). See, e.g., U.S. Ser. No. 60/435,001, which isincorporated herein by reference for all purposes.

In some embodiments of compounds of Formula I, II, or III, R₄ takentogether with R₁₂ forms an optionally substituted piperazine- ordiazepam of the formula:

R₃₁ and R₃₂ are independently chosen from hydrogen, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted aralkyl, and optionally substitutedheteroaralkyl; and n is 1 or 2. In some embodiments, R₃₁ is aryl (suchas phenyl), substituted aryl (such as lower alkyl-, lower alkoxy-,and/or halo-substituted phenyl), aralkyl (such as benzyl andphenylvinyl), heteroaralkyl, substituted aralkyl (such as substitutedbenzyl and substituted phenylvinyl), or substituted heteroaralkyl; R₃₂is hydrogen; and n is 1. See, e.g., PCT/US03/026093, which isincorporated herein by reference.R₄

When considering compounds of Formula I, in some embodiments, R₄ ischosen from hydrogen, optionally substituted C₁-C₁₃ alkyl, optionallysubstituted aryl, optionally substituted aryl-C₁-C₄-alkyl-, optionallysubstituted heterocyclyl, and optionally substitutedheteroaryl-C₁-C₄-alkyl- (such as hydrogen or optionally substitutedC₁-C₁₃ alkyl).

In some embodiments, R₄ is chosen from hydrogen, C₁-C₄ alkyl;cyclohexyl; phenyl substituted with hydroxyl, C₁-C₄ alkoxy or C₁-C₄alkyl; benzyl; and R₁₆-alkylene-, wherein R₁₆ is hydroxyl, carboxy,(C₁-C₄ alkoxy)carbonyl-, di(C₁-C₄ alkyl)amino-, (C₁-C₄ alkyl)amino-,amino, (C₁-C₄ alkoxy)carbonylamino-, C₁-C₄ alkoxy-, or optionallysubstituted N-heterocyclyl- (such as azetidinyl, morpholinyl, pyridinyl,indolyl, furanyl, pyrrolidinyl, piperidinyl or imidazolyl, each of whichmay be optionally substituted).

In some embodiments, R₄ is chosen from hydrogen, methyl, ethyl, propyl,butyl, cyclohexyl, carboxyethyl, carboxymethyl, methoxyethyl,hydroxyethyl, hydroxypropyl, dimethylaminoethyl, dimethylaminopropyl,diethylaminoethyl, diethylaminopropyl, aminopropyl, methylaminopropyl,2,2-dimethyl-3-(dimethylamino)propyl, aminoethyl, aminobutyl,aminopentyl, aminohexyl, isopropylaminopropyl, diisopropylaminoethyl,1-methyl-4-(diethylamino)butyl, (t-Boc)aminopropyl, hydroxyphenyl,benzyl, methoxyphenyl, methylmethoxyphenyl, dimethylphenyl, tolyl,ethylphenyl, (oxopyrrolidinyl)propyl, (methoxycarbonyl)ethyl,benzylpiperidinyl, pyridinylethyl, pyridinylmethyl, morpholinylethylmorpholinylpropyl, piperidinyl, azetidinylmethyl, azetidinylethyl,azetidinylpropyl pyrrolidinylethyl, pyrrolidinylpropyl,piperidinylmethyl, piperidinylethyl, imidazolylpropyl, imidazolylethyl,(ethylpyrrolidinyl)methyl, (methylpyrrolidinyl)ethyl,(methylpiperidinyl)propyl, (methylpiperazinyl)propyl, furanylmethyl andindolylethyl.

In some embodiments, R₄ is R₁₆-alkylene-, wherein R₁₆ is amino, C₁-C₄alkylamino-, di(C₁-C₄ alkyl)amino-, C₁-C₄ alkoxy-, hydroxyl, orN-heterocyclyl. In some embodiments, R₁₆ is amino. In some embodiments,the alkylene moiety of R₁₆-alkylene- has from 1 to 6 carbon atoms.

In some embodiments, R₄ is aminoethyl, aminopropyl, aminobutyl,aminopentyl, aminohexyl, methylaminoethyl, methylaminopropyl,methylaminobutyl, methylaminopentyl, methylaminohexyl,dimethylaminoethyl, dimethylaminopropyl, dimethylaminobutyl,dimethylaminopentyl, dimethylaminohexyl, ethylaminoethyl,ethylaminopropyl, ethylaminobutyl, ethylaminopentyl, ethylaminohexyl,diethylaminoethyl, diethylaminopropyl, diethylaminobutyyl,diethylaminopentyl, or diethylaminohexyl. In some embodiments, R₄ isaminopropyl.

R₁₂

When considering the compounds of Formula I, in some embodiments R₁₂ ischosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-,—C(O)—R₃, and —S(O)₂-R_(3a). In some embodiments, R₁₂ is optionallysubstituted C₁-C₁₃ alkyl (such as substituted C₁-C₄ alkyl); optionallysubstituted aralkyl (such as optionally substituted benzyl ornaphthylmethyl-); and optionally substituted heteroaralkyl. In someembodiments, R₁₂ is benzyl or benzyl substituted with one or more of thefollowing groups: carboxy, alkoxycarbonyl cyano, halo, C₁-C₄ alkyl-,C₁-C₄ alkoxy, nitro, methylenedioxy, or trifluoromethyl. In someembodiments, and as described below, R₁₂ is —C(O)R₃. In someembodiments, and as described below R₁₂ is —SO₂R_(3a).

R₁₂ is —C(O)R₃

When considering the compounds of Formula I, in some embodiments whenR₁₂ is —C(O)R₃, R₃ is selected from optionally substituted C₁-C₈ alkyl,optionally substituted aryl-C₁-C₄-alkyl-, optionally substitutedheteroaryl-C₁-C₄-alkyl-, optionally substituted heteroaryl, optionallysubstituted aryl, R₁₅O— and R₁₇—NH—, R₁₅ is chosen from optionallysubstituted C₁-C₈ alkyl and optionally substituted aryl, and R₁₇ ischosen from hydrogen, optionally substituted C₁-C₈ alkyl and optionallysubstituted aryl.

In some embodiments, R₃ is chosen from optionally substituted C₁-C₈alkyl, optionally substituted aryl-C₁-C₄-alkyl-, optionally substitutedheteroaryl-C₁-C₄-alkyl-, optionally substituted heteroaryl, andoptionally substituted aryl. In some embodiments, R₃ is chosen from

-   -   phenyl;    -   phenyl substituted with one or more of the following        substituents: halo; C₁-C₄ alkyl; C₁-C₄ alkyl substituted with        hydroxy (e.g., hydroxymethyl); C₁-C₄ alkoxy; C₁-C₄ alkyl        substituted with C₁-C₄ alkoxy, halo, nitro, formyl, carboxy,        cyano, methylenedioxy, ethylenedioxy, acyl (e.g., acetyl),        —N-acyl (e.g., N-acetyl) or trifluoromethyl;    -   benzyl;    -   phenoxymethyl-;    -   halophenoxymethyl-;    -   phenylvinyl-;    -   heteroaryl-;    -   heteroaryl- substituted with C₁-C₄ alkyl or C₁-C₄ alkyl        substituted with halo (e.g., CF₃);    -   C₁-C₄ alkyl substituted with C₁-C₄ alkoxy-; and    -   benzyloxymethyl-.

In some embodiments, when R₃ is not R₁₇NH— or R₁₅O—, R₃ is chosen fromphenyl, halophenyl, dihalophenyl, cyanophenyl,halo(trifluoromethyl)phenyl, hydroxymethylphenyl, methoxymethylphenyl,methoxyphenyl, ethoxyphenyl, carboxyphenyl, formylphenyl, ethylphenyl,tolyl, methylenedioxyphenyl, ethylenedioxyphenyl, methoxychlorophenyl,dihydro-benzodioxinyl, methylhalophenyl, trifluoromethylphenyl, furanyl,C₁-C₄ alkyl substituted furanyl, trifluoromethylfuranyl, C₁-C₄ alkylsubstituted trifluoromethylfuranyl, benzofuranyl, thiophenyl, C₁-C₄alkyl substituted thiophenyl, benzothiophenyl, benzothiadiazolyl,pyridinyl, indolyl, methylpyridinyl, trifluoromethylpyridinyl, pyrrolyl,quinolinyl, picolinyl, pyrazolyl, C₁-C₄ alkyl substituted pyrazolyl,N-methyl pyrazolyl, C₁-C₄ alkyl substituted N-methyl pyrazolyl, C₁-C₄alkyl substituted pyrazinyl, C₁-C₄ alkyl substituted isoxazolyl,benzoisoxazolyl, morpholinomethyl, methylthiomethyl, methoxymethyl,N-methyl imidazolyl, and imidazolyl. In some embodiments, R₃ isoptionally substituted phenyl (such as tolyl, halophenyl,methylhalophenyl, hydroxymethylphenyl, halo(trifluoromethyl)phenyl-,methylenedioxyphenyl, formylphenyl or cyanophenyl).

In some embodiments, when R₃ is R₁₇NH—, R₁₇ is chosen from hydrogen,C₁-C₄ alkyl; cyclohexyl; phenyl; and phenyl substituted with halo, C₁-C₄alkyl, trifluoromethyl, C₁-C₄ alkoxy, or C₁-C₄ alkylthio.

In some embodiments, when R₃ is R₁₇NH—, R₁₇ is hydrogen isopropyl,butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyl, methoxyphenyl,ethylphenyl, tolyl, trifluoromethylphenyl or methylthiophenyl.

In some embodiments, wherein R₃ is R₁₅O—, R₁₅ is chosen from optionallysubstituted C₁-C₈ alkyl and optionally substituted aryl.

R₁₂ is —SO₂R_(3a)

When considering the compounds of Formula I, in some embodiments, whenR₁₂ is —SO₂R_(3a), R_(3a) is chosen from C₁-C₁₃ alkyl-; phenyl-;naphthyl; phenyl substituted with cyano, halo, lower-alkyl-,lower-alkoxy, nitro, methylenedioxy, or trifluoromethyl-; biphenylyl andheteroaryl-. In some embodiments, R_(3a) is chosen from phenylsubstituted with halo, lower-alkyl-, lower-alkoxy, cyano, nitro,methlenedioxy, or trifluoromethyl-; and naphthyl-.

Salt Forms

Compounds of the invention will generally be capable of forming acidaddition salts (i.e., will comprise a site which reacts with apharmaceutically acceptable acid to form an acid addition salt.) Thepresent invention includes pharmaceutically acceptable acid additionsalts of the compounds of Formula I. Acid addition salts of the presentcompounds are prepared in a standard manner in a suitable solvent fromthe parent compound and an excess of an acid, such as hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic ormethanesulfonic.

The salts and/or solvates of the compounds of the Formula I which arenot pharmaceutically acceptable may be useful as intermediates in thepreparation of pharmaceutically acceptable salts and/or solvates ofcompounds of Formula I or the compounds of the Formula I themselves, andas such form another aspect of the present invention.

Compounds of Formula I

When considering the compounds of Formula I, in some embodiments,

-   -   one of W, X, Y, and Z is N and the others are C;    -   R₁ is benzyl, halobenzyl, methoxybenzyl-, cyanobenzyl, or        naphthalenylmethyl-; R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   T and T′ are independently a covalent bond or optionally        substituted lower alkylene (such as where T and T′ are both        absent);    -   R₁₂ is —C(O)R₃ wherein R₃ is optionally substituted C₁-C₈ alkyl        (e.g., C₁-C₈ alkyl substituted with lower-alkoxy), optionally        substituted heteroaryl, and optionally substituted aryl (such as        halophenyl, halomethylphenyl-, methylenedioxyphenyl-,        methoxyphenyl-, ethoxyphenyl-, cyanophenyl- or phenyl        substituted with lower-acyl or lower-alkylaminocarbonyl-, e.g.        methylaminocarbonyl- or ethylaminocarbonyl-, or        di(lower-alkyl)aminocarbonyl-, e.g. dimethylaminocarbonyl- or        diethylaminocarbonyl- or lower alkyl); and    -   R₄is R₁₆-alkylene-, wherein R₁₆ is amino, C₁-C₄ alkylamino-,        di(C₁-C₄ alkyl)amino-, C₁-C₄ alkoxy-, hydroxyl, or        N-heterocyclyl.

In some embodiments, R₁ is benzyl, halobenzyl, methoxybenzyl,cyanobenzyl, or naphthalenylmethyl; and R₂ is propyl (such as i- orc-propyl).

In some embodiments,

-   -   one of W, X, Y, and Z is N and the others are C;    -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl; R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   T and T′ are independently a covalent bond or optionally        substituted lower alkylene (such as where T and T′ are both        absent); and    -   R₄ taken together with R₁₂ form an optionally substituted        imidazolyl.

In some embodiments,

-   -   one of W, X, Y, and Z is N and the others are C;    -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl; R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   T and T′ are independently a covalent bond or optionally        substituted lower alkylene (such as where T and T′ are both        absent); and    -   R₄ taken together with R₁₂ form an optionally substituted        imidazolidinyl ring.

In some embodiments,

-   -   one of W, X, Y, and Z is N and the others are C;    -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   T and T′ are independently a covalent bond or optionally        substituted lower alkylene (such as where T and T′ are both        absent); and    -   R₄ taken together with R₁₂ form an optionally substituted        piperazinyl ring.

In some embodiments,

-   -   one of W, X, Y, and Z is N and the others are C;    -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   T and T′ are independently a covalent bond or optionally        substituted lower alkylene (such as where T and T′ are both        absent); and    -   R₄ taken together with R₁₂ form an optionally substituted        diazepinoyl ring.        Compounds of Formula II

When considering the compounds of Formula II, in some embodiments,

-   -   R₁ is benzyl, halobenzyl, methoxybenzyl-, cyanobenzyl, or        naphthalenylmethyl-;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   one of T and T′ is absent and the other is optionally        substituted alkylene;    -   R₁₂ is —C(O)R₃ wherein R₃ is optionally substituted C₁-C₈ alkyl        (e.g., C₁-C₈ alkyl substituted with lower-alkoxy), optionally        substituted heteroaryl, and optionally substituted aryl (such as        halophenyl, halomethylphenyl-, methylenedioxyphenyl-,        methoxyphenyl-, ethoxyphenyl-, cyanophenyl- or phenyl        substituted with lower-acyl or lower-alkylaminocarbonyl-, e.g.        methylaminocarbonyl- or ethylaminocarbonyl-, or        di(lower-alkyl)aminocarbonyl-, e.g. dimethylaminocarbonyl- or        diethylaminocarbonyl- or lower alkyl); and R₄ is R₁₆-alkylene-,        wherein R₁₆ is amino, C₁-C₄ alkylamino-, di(C₁-C₄ alkyl)amino-,        C₁-C₄ alkoxy-, hydroxyl, or N-heterocyclyl.

In some embodiments, R₁ is benzyl, halobenzyl, methoxybenzyl,cyanobenzyl, or naphthalenylmethyl; and R₂ is propyl (such as i- orc-propyl).

In some embodiments,

-   -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   one of T and T′ is absent and the other is optionally        substituted alkylene; and    -   R₄ taken together with R₁₂ form an optionally substituted        imidazolyl.

In some embodiments,

-   -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   one of T and T′ is absent and the other is optionally        substituted alkylene; and    -   R₄ taken together with R₁₂ form an optionally substituted        imidazolidinyl ring.

In some embodiments,

-   -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   one of T and T′ is absent and the other is optionally        substituted alkylene; and    -   R₄ taken together with R₁₂ form an optionally substituted        piperazinyl ring.

In some embodiments,

-   -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano;    -   one of T and T′ is absent and the other is optionally        substituted alkylene; and    -   R₄ taken together with R₁₂ form an optionally substituted        diazepinoyl ring.        Compounds of Formula III

When considering the compounds of Formula III, in some embodiments,

-   -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R₂ is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano; and    -   R₄ taken together with R₁₂ form an optionally substituted        piperazinyl ring.

In some embodiments,

-   -   R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or        naphthalenylmethyl;    -   R₂ is optionally substituted C₁-C₄ alkyl;    -   R_(2′) is hydrogen;    -   R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,        amino, alkylamino, dialkylamino, hydroxyl, halogen (such as        chloro and fluoro), C₁-C₄ alkyl (such as methyl), C₁-C₄        haloalkyl (such as trifluoromethyl), C₁-C₄ alkoxy (such as        methoxy), C₁-C₄ haloalkoxy and cyano; and    -   R₄ taken together with R₁₂ form an optionally substituted        diazepinoyl ring.

Once made, the compounds of the invention find use in a variety ofapplications involving alteration of mitosis. As will be appreciated bythose skilled in the art, mitosis may be altered in a variety of ways;that is, one can affect mitosis either by increasing or decreasing theactivity of a component in the mitotic pathway. Stated differently,mitosis may be affected (e.g., disrupted) by disturbing equilibrium,either by inhibiting or activating certain components. Similarapproaches may be used to alter meiosis.

In some embodiments, the compounds of the invention are used to inhibitmitotic spindle formation, thus causing prolonged cell cycle arrest inmitosis. By “inhibit” in this context is meant decreasing or interferingwith mitotic spindle formation or causing mitotic spindle dysfunction.By “mitotic spindle formation” herein is meant organization ofmicrotubules into bipolar structures by mitotic kinesins. By “mitoticspindle dysfunction” herein is meant mitotic arrest and monopolarspindle formation.

The compounds of the invention are useful to bind to, and/or inhibit theactivity of, a mitotic kinesin, KSP. In some embodiments, the KSP ishuman KSP, although the compounds may be used to bind to or inhibit theactivity of KSP kinesins from other organisms. In this context,“inhibit” means either increasing or decreasing spindle pole separation,causing malformation, i.e., splaying, of mitotic spindle poles, orotherwise causing morphological perturbation of the mitotic spindle.Also included within the definition of KSP for these purposes arevariants and/or fragments of KSP. See U.S. Pat. No. 6,437,115, herebyincorporated by reference in its entirety. The compounds of theinvention have been shown to have specificity for KSP. However, thepresent invention includes the use of the compounds to bind to ormodulate other mitotic kinesins.

The compounds of the invention are used to treat cellular proliferationdiseases. Such disease states which can be treated by the compounds,compositions and methods provided herein include, but are not limitedto, cancer (further discussed below), autoimmune disease, fungaldisorders, arthritis, graft rejection, inflammatory bowel disease,cellular proliferation induced after medical procedures, including, butnot limited to, surgery, angioplasty, and the like. Treatment includesinhibiting cellular proliferation. It is appreciated that in some casesthe cells may not be in an abnormal state and still require treatment.Thus, in some embodiments, the invention herein includes application tocells or individuals afflicted or subject to impending affliction withany one of these disorders or states.

The compounds, compositions and methods provided herein are useful forthe treatment of cancer including solid tumors such as skin, breast,brain, cervical carcinomas, testicular carcinomas, etc. In someembodiments, cancers that may be treated by the compounds, compositionsand methods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor(nephroblastoma), lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma (pinealoma), glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia (acuteand chronic), acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignantlymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above identified conditions.

For assay of KSP-modulating activity, generally either KSP or a compoundaccording to the invention is non-diffusably bound to an insolublesupport having isolated sample receiving areas (e.g., a microtiterplate, an array, etc.). The insoluble support may be made of anymaterial to which the sample can be bound, is readily separated fromsoluble material, and is otherwise compatible with the overall method ofscreening. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes and beads. These are typically madeof glass, plastic (e.g., polystyrene), polysaccharides, nylon ornitrocellulose, Teflon™, etc. Microtiter plates and arrays areconvenient because a large number of assays can be carried outsimultaneously, using small amounts of reagents and samples. The mannerof binding of the sample is not crucial so long as it is compatible withthe reagents and overall methods of the invention, maintains theactivity of the sample and is nondiffusable. Methods of binding includethe use of antibodies (which do not sterically block either the ligandbinding site or activation sequence when the protein is bound to thesupport), direct binding to “sticky” or ionic supports, chemicalcrosslinking, the synthesis of the protein or agent on the surface, etc.Following binding of the sample, excess unbound material is removed bywashing. The sample receiving areas may then be blocked throughincubation with bovine serum albumin (BSA), casein or other innocuousprotein or other moiety.

The compounds of the invention may be used on their own to inhibit theactivity of a mitotic kinesin, such as KSP. In some embodiments, acompound of the invention is combined with KSP and the activity of KSPis assayed. Kinesin (including KSP) activity is known in the art andincludes one or more kinesin activities. Kinesin activities include theability to affect ATP hydrolysis; microtubule binding; gliding andpolymerization/depolymerization (effects on microtubule dynamics);binding to other proteins of the spindle; binding to proteins involvedin cell-cycle control; serving as a substrate to other enzymes, such askinases or proteases; and specific kinesin cellular activities such asspindle pole separation.

Methods of performing motility assays are well known to those of skillin the art. (See e.g., Hall, et al. (1996), Biophys. J., 71: 3467-3476,Turner et al., 1996, AnaL Biochem. 242 (1):20-5; Gittes et al., 1996,Biophys. J. 70(1): 418-29; Shirakawa et al., 1995, J. Exp. BioL 198:1809-15; Winkelmann et al., 1995, Biophys. J. 68: 2444-53; Winkelmann etal., 1995, Biophys. J. 68: 72S.)

Methods known in the art for determining ATPase hydrolysis activity alsocan be used. Suitably, solution based assays are utilized. U.S. Pat. No.6,410,254, hereby incorporated by reference in its entirety, describessuch assays. Alternatively, conventional methods are used. For example,Pi release from kinesin can be quantified. In some embodiments, theATPase hydrolysis activity assay utilizes 0.3 M PCA (perchloric acid)and malachite green reagent (8.27 mM sodium molybdate II, 0.33 mMmalachite green oxalate, and 0.8 mM Triton X-100). To perform the assay,10 μL of the reaction mixture is quenched in 90 μL of cold 0.3 M PCA.Phosphate standards are used so data can be converted to mM inorganicphosphate released. When all reactions and standards have been quenchedin PCA, 100 μL of malachite green reagent is added to the relevant wellsin e.g., a microtiter plate. The mixture is developed for 10-15 minutesand the plate is read at an absorbance of 650 nm. If phosphate standardswere used, absorbance readings can be converted to mM P_(i) and plottedover time. Additionally, ATPase assays known in the art include theluciferase assay.

ATPase activity of kinesin motor domains also can be used to monitor theeffects of agents and are well known to those skilled in the art. Insome embodiments ATPase assays of kinesin are performed in the absenceof microtubules. In some embodiments, the ATPase assays are performed inthe presence of microtubules. Different types of agents can be detectedin the above assays. In some embodiments, the effect of an agent isindependent of the concentration of microtubules and ATP. In someembodiments, the effect of the agents on kinesin ATPase can be decreasedby increasing the concentrations of ATP, microtubules or both. In someembodiments, the effect of the agent is increased by increasingconcentrations of ATP, microtubules or both.

Compounds that inhibit the biochemical activity of KSP in vitro may thenbe screened in vivo. In vivo screening methods include assays of cellcycle distribution, cell viability, or the presence, morphology,activity, distribution, or number of mitotic spindles. Methods formonitoring cell cycle distribution of a cell population, for example, byflow cytometry, are well known to those skilled in the art, as aremethods for determining cell viability. See for example, U.S. Pat. No.6,437,115, hereby incorporated by reference in its entirety. Microscopicmethods for monitoring spindle formation and malformation are well knownto those of skill in the art (see, e.g., Whitehead and Rattner (1998),J. Cell Sci. 111:2551-61; Galgio et al, (1996) J. Cell Biol.,135:399-414), each incorporated herein by reference in its entirety.

The compounds of the invention inhibit the KSP kinesin. One measure ofinhibition is IC₅₀, defined as the concentration of the compound atwhich the activity of KSP is decreased by fifty percent relative to acontrol. In some embodiments, compounds have IC₅₀'s of less than about 1mM. In some embodiments, compounds have IC₅₀'s of less than about 100μM. In some embodiments, compounds have IC₅₀'s of less than about 10 μM.In some embodiments, compounds have IC₅₀'s of less than about 1 μM. Insome embodiments, compounds have IC₅₀'s of less than about 100 nM. Insome embodiments, compounds have IC₅₀'s of less than about 10 nM.Measurement of IC₅₀ is done using an ATPase assay such as describedherein.

Another measure of inhibition is K_(i). For compounds with IC₅₀'s lessthan 1 μM, the K_(i) or K_(d) is defined as the dissociation rateconstant for the interaction of the compounds described herein with KSP.In some embodiments, compounds have compounds have K_(i)'s of less thanabout 100 μM. In some embodiments, compounds have K_(i)'s of less thanabout 10 μM. In some embodiments, compounds have K_(i)'s of less thanabout 1 μM. In some embodiments, compounds have K_(i)'s of less thanabout 100 nM. In some embodiments, compounds have K_(i)'s of less thanabout 10 nM.

The K_(i) for a compound is determined from the IC₅₀ based on threeassumptions and the Michaelis-Menten equation. First, only one compoundmolecule binds to the enzyme and there is no cooperativity. Second, theconcentrations of active enzyme and the compound tested are known (i.e.,there are no significant amounts of impurities or inactive forms in thepreparations). Third, the enzymatic rate of the enzyme-inhibitor complexis zero. The rate (i.e., compound concentration) data are fitted to theequation:$V = {V_{\max}{E_{0}\lbrack {I - \frac{( {E_{0} + I_{0} + {Kd}} ) - \sqrt{( {E_{0} + I_{0} + {Kd}} )^{2} - {4E_{0}I_{0}}}}{2E_{0}}} \rbrack}}$where V is the observed rate, V_(max) is the rate of the free enzyme, I₀is the inhibitor concentration, E₀ is the enzyme concentration, andK_(d) is the dissociation constant of the enzyme-inhibitor complex.

Another measure of inhibition is GI₅₀, defined as the concentration ofthe compound that results in a decrease in the rate of cell growth byfifty percent. In some embodiments, compounds have GI₅₀'s of less thanabout 1 mM. In some embodiments, compounds have a GI₅₀ of less thanabout 20 μM. In some embodiments, compounds have a GI₅₀ of less thanabout 10 μM. In some embodiments, compounds have a GI₅₀ of less thanabout 1 μM. In some embodiments, compounds have a GI₅₀ of less thanabout 100 nM. In some embodiments, compounds have a GI₅₀ of less thanabout 10 nM. Measurement of GI₅₀ is done using a cell proliferationassay such as described herein. Compounds of this class were found toinhibit cell proliferation.

In vitro potency of small molecule inhibitors is determined, forexample, by assaying human ovarian cancer cells (SKOV3) for viabilityfollowing a 72-hour exposure to a 9-point dilution series of compound.Cell viability is determined by measuring the absorbance of formazon, aproduct formed by the bioreduction of MTS/PMS, a commercially availablereagent. Each point on the dose-response curve is calculated as apercent of untreated control cells at 72 hours minus backgroundabsorption (complete cell kill).

Anti-proliferative compounds that have been successfully applied in theclinic to treatment of cancer (cancer chemotherapeutics) have GI₅₀'sthat vary greatly. For example, in A549 cells, paclitaxel GI₅₀ is 4 nM,doxorubicin is 63 nM, 5-fluorouracil is 1 μM, and hydroxyurea is 500 μM(data provided by National Cancer Institute, Developmental TherapeuticProgram, http://dtp.nci.nih.gov/). Therefore, compounds that inhibitcellular proliferation, irrespective of the concentration demonstratinginhibition, have potential clinical usefulness.

To employ the compounds of the invention in a method of screening forcompounds that bind to KSP kinesin, the KSP is bound to a support, and acompound of the invention is added to the assay. Alternatively, thecompound of the invention is bound to the support and KSP is added.Classes of compounds among which novel binding agents may be soughtinclude specific antibodies, non-natural binding agents identified inscreens of chemical libraries, peptide analogs, etc. In someembodiments, candidate agents are screened a low toxicity for humancells. A wide variety of assays may be used for this purpose, includinglabeled in vitro protein-protein binding assays, electrophoreticmobility shift assays, immunoassays for protein binding, functionalassays (phosphorylation assays, etc.) and the like.

The determination of the binding of the compound of the invention to KSPmay be done in a number of ways. In some embodiments, the compound islabeled, for example, with a fluorescent or radioactive moiety, andbinding is determined directly. For example, this may be done byattaching all or a portion of KSP to a solid support, adding a labeledtest compound (for example a compound of the invention in which at leastone atom has been replaced by a detectable isotope), washing off excessreagent, and determining whether the amount of the label is that presenton the solid support.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the kinesin proteins may be labeled at tyrosine positions using ¹²⁵I, orwith fluorophores. Alternatively, more than one component may be labeledwith different labels; using ¹²⁵I for the proteins, for example, and afluorophor for the antimitotic agents.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates. “Candidate agent” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for bioactivity. They may be capableof directly or indirectly altering the cellular proliferation phenotypeor the expression of a cellular proliferation sequence, including bothnucleic acid sequences and protein sequences. In other cases, alterationof cellular proliferation protein binding and/or activity is screened.Screens of this sort may be performed either in the presence or absenceof microtubules. In the case where protein binding or activity isscreened, some embodiments exclude molecules already known to bind tothat protein, for example, polymer structures such as microtubules, andenergy sources such as ATP. Some embodiments of assays herein includecandidate agents which do not bind the cellular proliferation protein inits endogenous native state termed herein as “exogenous” agents. In someembodiments, exogenous agents further exclude antibodies to KSP.

Candidate agents can encompass numerous chemical classes, thoughtypically they are organic molecules, such as small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, such as hydrogen bonding andlipophilic binding, and typically include at least an amine, carbonyl-,hydroxyl-, ether, or carboxyl group, often, at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, and/or amidification to producestructural analogs.

Competitive screening assays may be done by combining KSP and a drugcandidate in a first sample. A second sample comprises a compound of thepresent invention, KSP and a drug candidate. This may be performed ineither the presence or absence of microtubules. The binding of the drugcandidate is determined for both samples, and a change, or difference inbinding between the two samples indicates the presence of a drugcandidate capable of binding to KSP and potentially inhibiting itsactivity. That is, if the binding of the drug candidate is different inthe second sample relative to the first sample, the drug candidate iscapable of binding to KSP.

In some embodiments, the binding of the candidate agent to KSP isdetermined through the use of competitive binding assays. In someembodiments, the competitor is a binding moiety known to bind to KSP,such as an antibody, peptide, binding partner, ligand, etc. Undercertain circumstances, there may be competitive binding as between thecandidate agent and the binding moiety, with the binding moietydisplacing the candidate agent.

In some embodiments, the candidate agent is labeled. Either thecandidate agent, or the competitor, or both, is added first to KSP for atime sufficient to allow binding, if present. Incubations may beperformed at any temperature which facilitates optimal activity,typically between 4 and 40° C.

Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high throughput screening. Typicallybetween 0.1 and 1 hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In some embodiments, the competitor is added first, followed by thecandidate agent. Displacement of the competitor is an indication thecandidate agent is binding to KSP and thus is capable of binding to, andpotentially inhibiting, the activity of KSP. In some embodiments, eithercomponent can be labeled. Thus, for example, if the competitor islabeled, the presence of label in the wash solution indicatesdisplacement by the agent. Alternatively, if the candidate agent islabeled, the presence of the label on the support indicatesdisplacement.

In some embodiments, the candidate agent is added first, with incubationand washing, followed by the competitor. The absence of binding by thecompetitor may indicate the candidate agent is bound to KSP with ahigher affinity. Thus, if the candidate agent is labeled, the presenceof the label on the support, coupled with a lack of competitor binding,may indicate the candidate agent is capable of binding to KSP.

Inhibition is tested by screening for candidate agents capable ofinhibiting the activity of KSP comprising the steps of combining acandidate agent with KSP, as above, and determining an alteration in thebiological activity of KSP. In some embodiments, the candidate agentshould both bind to KSP (although this may not be necessary), and alterits biological or biochemical activity as defined herein. The methodsinclude both in vitro screening methods and in vivo screening of cellsfor alterations in cell cycle distribution, cell viability, or for thepresence, morpohology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

Alternatively, differential screening may be used to identify drugcandidates that bind to the native KSP, but cannot bind to modified KSP.

Positive controls and negative controls may be used in the assays.Suitably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Accordingly, the compounds of the invention are administered to cells.By “administered” herein is meant administration of a therapeuticallyeffective dose of a compound of the invention to a cell either in cellculture or in a patient. By “therapeutically effective dose” herein ismeant a dose that produces the effects for which it is administered. Theexact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for systemic versus localized delivery,age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art. By “cells” herein is meant any cell in which mitosis or meiosiscan be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, such as mammals, and other organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In some embodiments the patient is a mammal. In someembodiments, the patient is human.

Compounds of the invention having the desired pharmacological activitymay be administered, generally as a pharmaceutically acceptablecomposition comprising an pharmaceutical excipient, to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %.

The agents may be administered alone or in combination with othertreatments, i.e., radiation, or other chemotherapeutic agents such asthe taxane class of agents that appear to act on microtubule formationor the camptothecin class of topoisomerase I inhibitors. When used,other chemotherapeutic agents may be administered before, concurrently,or after administration of a compound of the present invention. In oneaspect of the invention, a compound of the present invention isco-administered with one or more other chemotherapeutic agents. By“co-administer” it is meant that the present compounds are administeredto a patient such that the present compounds as well as theco-administered compound may be found in the patient's bloodstream atthe same time, regardless when the compounds are actually administered,including simultaneously.

The administration of the compounds and compositions of the presentinvention can be done in a variety of ways, including, but not limitedto, orally, subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the compound or composition may be directlyapplied as a solution or spray.

Pharmaceutical dosage forms include a compound of Formula I, II, or IIIor a pharmaceutically acceptable salt, solvate, or solvate of a saltthereof, and one or more pharmaceutical excipients. As is known in theart, pharmaceutical excipients are secondary ingredients which functionto enable or enhance the delivery of a drug or medicine in a variety ofdosage forms (e.g.: oral forms such as tablets, capsules, and liquids;topical forms such as dermal, opthalmic, and otic forms; suppositories;injectables; respiratory forms and the like). Pharmaceutical excipientsinclude inert or inactive ingredients, synergists or chemicals thatsubstantively contribute to the medicinal effects of the activeingredient. For example, pharmaceutical excipients may function toimprove flow characteristics, product uniformity, stability, taste, orappearance, to ease handling and administration of dose, for convenienceof use, or to control bioavailability. While pharmaceutical excipientsare commonly described as being inert or inactive, it is appreciated inthe art that there is a relationship between the properties of thepharmaceutical excipients and the dosage forms containing them.

Pharmaceutical excipients suitable for use as carriers or diluents arewell known in the art, and may be used in a variety of formulations.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, Editor, Mack Publishing Company (1990); Remington: The Scienceand Practice of Pharmacy, 20th Edition, A. R. Gennaro, Editor,Lippincott Williams & Wilkins (2000); Handbook of PharmaceuticalExcipients, 3rd Edition, A. H. Kibbe, Editor, American PharmaceuticalAssociation, and Pharmaceutical Press (2000); and Handbook ofPharmaceutical Additives, compiled by Michael and Irene Ash,Gower(1995), each of which is incorporated herein by reference for allpurposes.

Oral solid dosage forms such as tablets will typically comprise one ormore pharmaceutical excipients, which may for example help impartsatisfactory processing and compression characteristics, or provideadditional desirable physical characteristics to the tablet. Suchpharmaceutical excipients may be selected from diluents, binders,glidants, lubricants, disintegrants, colors, flavors, sweetening agents,polymers, waxes or other solubility-retarding materials.

Compositions for intravenous administration will generally compriseintravenous fluids, i.e., sterile solutions of simple chemicals such assugars, amino acids or electrolytes, which can be easily carried by thecirculatory system and assimilated. Such fluids are prepared with waterfor injection USP.

Fluids used commonly for intravenous (IV) use are disclosed inRemington, the Science and Practice of Pharmacy [full citationpreviously provided], and include:

-   -   alcohol (e.g., in dextrose and water (“D/W”) [e.g., 5% dextrose]        or dextrose and water [e.g., 5% dextrose] in normal saline        solution (“NSS”); e.g. 5% alcohol);    -   synthetic amino acid such as Aminosyn, FreAmine, Travasol, e.g.,        3.5 or 7; 8.5; 3.5, 5.5 or 8.5% respectively;    -   ammonium chloride e.g., 2.14%;    -   dextran 40, in NSS e.g., 10% or in D5/W e.g., 10%;    -   dextran 70, in NSS e.g., 6% or in D5/W e.g., 6%;    -   dextrose (glucose, D5/W) e.g., 2.5-50%;    -   dextrose and sodium chloride e.g., 5-20% dextrose and 0.22-0.9%        NaCl;    -   lactated Ringer's (Hartmann's) e.g., NaCl 0.6%, KCl 0.03%, CaCl₂        0.02%;    -   lactate 0.3%;    -   mannitol e.g., 5%, optionally in combination with dextrose e.g.,        10% or NaCl e.g., 15 or 20%;    -   multiple electrolyte solutions with varying combinations of        electrolytes, dextrose, fructose, invert sugar Ringer's e.g.,        NaCl 0.86%, KCl 0.03%, CaCl₂ 0.033%;    -   sodium bicarbonate e.g., 5%;    -   sodium chloride e.g., 0.45, 0.9, 3, or 5%;    -   sodium lactate e.g., ⅙ M; and    -   sterile water for injection        The pH of such fluids may vary, and will typically be from 3.5        to 8 such as known in the art.

All references and publications, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference as if each individual reference andpublication were specifically and inidividually indicated to beincorporated by reference herein as though fully set forth.

It is to be understood that the present invention covers allcombinations of subgroups described herein above.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.

The following examples serve to more fully describe the manner of usingthe above-described invention. It is understood that these examples inno way serve to limit the true scope of this invention, but rather arepresented for illustrative purposes.

EXAMPLES Example 1

Inhibition of Cellular Viability in Tumor Cell Lines Treated with KSPInhibitors.r

Materials and Solutions:

-   Cells: SKOV3, Ovarian Cancer (human).-   Media: Phenol Red Free RPMI+5% Fetal Bovine Serum+2 mM L-glutamine.-   Colorimetric Agent for Determining Cell Viability: Promega MTS    tetrazolium compound.-   Control Compound for max cell kill: Topotecan, 1 μM.

Procedure: Day 1—Cell Plating:

Adherent SKOV3 cells are washed with 10 mLs of PBS followed by theaddition of 2 mLs of 0.25% trypsin and incubation for 5 minutes at 37°C. The cells are rinsed from the flask using 8 mL of media (phenolred-free RPMI+5% FBS) and transferred to fresh flask. Cell concentrationis determined using a Coulter counter and the appropriate volume ofcells to achieve 1000 cells/100 μL is calculated. 100 μL of media cellsuspension (adjusted to 1000 cells/100 μL) is added to all wells of96-well plates, followed by incubation for 18 to 24 hours at 37° C.,100% humidity, and 5% CO₂, allowing the cells to adhere to the plates.

Procedure: Day 2—Compound Addition:

To one column of the wells of an autoclaved assay block are added aninitial 2.5 μL of test compound(s) at 400× the highest desiredconcentration. 1.25 μL of 400× (400 μM) Topotecan is added to otherwells (optical density's from these wells are used to subtract out forbackground absorbance of dead cells and vehicle). 500 μL of mediawithout DMSO are added to the wells containing test compound, and 250 μLto the Topotecan wells. 250 μL of media+0.5% DMSO is added to allremaining wells, into which the test compound(s) are serially diluted.By row, compound-containing media is replica plated (in duplicate) fromthe assay block to the corresponding cell plates. The cell plates areincubated for 72 hours at 37° C., 100% humidity, and 5% CO₂.

Procedure: Day 4—MTS Addition and OD Reading:

The plates are removed from the incubator and 40 μl MTS/PMS is added toeach well. Plates are then incubated for 120 minutes at 37° C., 100%humidity, 5% CO₂, followed by reading the ODs at 490 nm after a 5 secondshaking cycle in a ninety-six well spectrophotometer.

Data Analysis

The normalized % of control (absorbance-background) is calculated and anXLfit is used to generate a dose-response curve from which theconcentration of compound required to inhibit viability by 50% isdetermined. The compounds of the present invention have activity whentested by this method as described above.

Example 2

Enantiomer Separation

In general, the procedures described above can be used to preparesubstantially pure or enriched R— or S-enantiomers by selected astarting amino acid of the appropriate R— or S-configuration. In someembodiments, compounds have an R-configuration at the stereogenic centerto which R₂ is attached. An R:S mixture can be separated into itsconstituent pure enantiomers by methods well known to those skilled inthe art. These include the formation and separation of diastereomericderivatives such as those formed by reaction with an optically pure acidsuch as dibenzoyltartaric acid. Alternatively, separation can beaccomplished by chiral chromatography, for example, using the followingconditions:

-   -   Column: Chiralcel OD 20×250 mm;    -   Sample loaded ˜100 mg mL⁻¹ in 1:2 ethanol:hexane containing        0.01% isopropylamine;    -   Chromatography conditions: isocratic elution with 1:2        ethanol:hexane containing 0.01% isopropylamine at a flow rate of        15 mL min⁻¹;    -   UV detection at 254 nm.

Example 3

Monopolar Spindle Formation Following Application of a KSP Inhibitor

Human tumor cells Skov-3 (ovarian) are plated in 96-well plates atdensities of 4,000 cells per well, allowed to adhere for 24 hours, andtreated with various concentrations of the pyridmidinone derivatives for24 hours. Cells are fixed in 4% formaldehyde and stained withantitubulin antibodies (subsequently recognized usingfluorescently-labeled secondary antibody) and Hoechst dye (which stainsDNA).

Visual inspection reveal that the compounds caused cell cycle arrest inthe prometaphase stage of mitosis. DNA is condensed and spindleformation is initiated, but arrested cells uniformly display monopolarspindles, indicating that there is an inhibition of spindle pole bodyseparation. Microinjection of anti-KSP antibodies also causes mitoticarrest with arrested cells displaying monopolar spindles.

Example 4

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated withKSP Inhibitors.

Cells are plated in 96-well plates at densities from 1000-2500cells/well of a 96-well plate and allowed to adhere/grow for 24 hours.They are then treated with various concentrations of drug for 48 hours.The time at which compounds are added is considered T₀. Atetrazolium-based assay using the reagent3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) (U.S. Pat. No. 5,185,450) (see Promega product catalog #G3580,CellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay) is usedto determine the number of viable cells at T₀ and the number of cellsremaining after 48 hours compound exposure. The number of cellsremaining after 48 hours is compared to the number of viable cells atthe time of drug addition, allowing for calculation of growthinhibition.

The growth over 48 hours of cells in control wells that are treated withvehicle only (0.25% DMSO) is considered 100% growth and the growth ofcells in wells with compounds is compared to this.

A Gi₅₀ is calculated by plotting the concentration of compound in μM vsthe percentage of cell growth in treated wells. The Gi₅₀ calculated forthe compounds is the estimated concentration at which growth isinhibited by 50% compared to control, i.e., the concentration at which:100×[(Treated₄₈ −T ₀)/(Control₄₈ −T ₀)]=50wherein Treated₄₈ is the value at 48 hours for the treated cells andControl₄₈ is the value at 48 hours for the control population.

All concentrations of compounds are tested in duplicate and controls areaveraged over 12 wells. A very similar 96-well plate layout and Gi₅₀calculation scheme is used by the National Cancer Institute (see Monks,et al., J. NatI. Cancer Inst. 83:757-766 (1991)). However, the method bywhich the National Cancer Institute quantitates cell number does not useMTS, but instead employs alternative methods.

Compounds of the invention inhibit cell proliferation in human ovariantumor cell lines (SKOV-3).

Example 5

Calculation of IC₅₀:

Measurement of a compound's IC₅₀ for KSP activity uses an ATPase assay.The following solutions are used: Solution 1 consists of 3 mMphosphoenolpyruvate potassium salt (Sigma P-7127), 2 mM ATP (SigmaA-3377), 1 mM IDTT (Sigma D-9779), 5 μM paclitaxel (Sigma T-7402), 10ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2mM MgCl2 (VWR JT400301), and 1 mM EGTA (Sigma E3889). Solution 2consists of 1 mM NADH (Sigma N8129), 0.2 mg/ml BSA (Sigma A7906),pyruvate kinase 7 U/ml, L-lactate dehydrogenase 10 U/ml (Sigma P0294),100 nM KSP motor domain, 50 μg/ml microtubules, 1 mM DTT (Sigma D9779),5 μM paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgCl2 (VWR JT4003-01), and 1 mMEGTA (Sigma E3889). Serial dilutions (8-12 two-fold dilutions) of thecompound are made in a 96-well microtiter plate (Coming Costar 3695)using Solution 1. Following serial dilution each well has 50 μl ofSolution 1. The reaction is started by adding 50 μl of solution 2 toeach well. This may be done with a multichannel pipettor either manuallyor with automated liquid handling devices. The microtiter plate is thentransferred to a microplate absorbance reader and multiple absorbancereadings at 340 nm are taken for each well in a kinetic mode. Theobserved rate of change, which is proportional to the ATPase rate, isthen plotted as a function of the compound concentration. For a standardIC₅₀ determination the data acquired is fit by the following fourparameter equation using a nonlinear fitting program (e.g., Grafit 4):$y = {\frac{Range}{1 + ( \frac{x}{{IC}_{50}} )^{S}} + {Background}}$where y is the observed rate and x is the compound concentration.

1. At least one chemical entity chosen from compounds of Formula I:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein, W, X, Y, and Z are independently —N═, N, —C═, CR₁₈, O or S andZ is optionally absent, provided that: the ring comprising W, X, Y, andoptionally Z is heterocyclic; no more than two of W, X, Y, and Z is —N═,and W, X, or Y can be O or S only when Z is absent; the dashed lines inthe structure depict optional double bonds; T and T′ are independently acovalent bond or optionally substituted lower alkylene; R₁ is chosenfrom hydrogen, optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-, optionally substitutedheteroaryl-, and optionally substituted heteroaralkyl-; R₂ and R_(2′)are independently chosen from hydrogen, optionally substituted alkyl-,optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaryl-, and optionally substitutedheteroaralkyl-; or R₂ and R_(2′) taken together form an optionallysubstituted 3- to 7-membered ring; R₁₂ is chosen from hydrogen,optionally substituted alkyl-, optionally substituted aryl-, optionallysubstituted aralkyl-, optionally substituted heteroaryl-, optionallysubstituted heteroaralkyl-, —C(O)—R₃, and —S(O)₂—R_(3a); R₃ is chosenfrom hydrogen, optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-, optionally substitutedheteroaryl-, optionally substituted heteroaralkyl-, R₁₅O— and R₁₇—NH—;R_(3a) is chosen from optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-, andR₁₇—NH—; R₄ is chosen from hydrogen, optionally substituted alkyl-,optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaralkyl-, and optionally substitutedheterocyclyl-; or R₄ taken together with R₁₂, and the nitrogen to whichthey are bound, form an optionally substituted 5- to 12-memberednitrogen-containing heterocycle, which optionally incorporates one ortwo additional heteroatoms, selected from N, O, and S in the heterocyclering; or R₄ taken together with R₂ form an optionally substituted 5- to12-membered nitrogen-containing heterocycle, which optionallyincorporates one or two additional heteroatoms, selected from N, O, andS in the heterocycle ring; R₅, R₆, R₇, R₈, and R₁₈ are independentlychosen from hydrogen, acyl, optionally substituted alkyl-, optionallysubstituted alkoxy, halogen, hydroxyl, nitro, cyano, optionallysubstituted amino, alkylsulfonyl-, alkylsulfonamido-, alkylthio-,carboxyalkyl-, carboxamido-, aminocarbonyl-, optionally substituted aryland optionally substituted heteroaryl-, provided that R₅, R₆, R₇ and R₈is absent where W, X, Y, or Z, respectively, is —N═, O, S or absent; R₁₅is chosen from optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-, optionally substitutedheteroaryl-, and optionally substituted heteroaralkyl-; and R₁₇ ishydrogen, optionally substituted alkyl, optionally substituted aryl,optionally substituted aralkyl, optionally substituted heteroaryl, oroptionally substituted heteroaralkyl-.
 2. At least one chemical entitychosen from compounds of Formula II:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein T and T′ are independently a covalent bond or optionallysubstituted lower alkylene, provided that T and T′ are not both covalentbonds; R₁ is chosen from hydrogen, optionally substituted alkyl-,optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaryl-, and optionally substitutedheteroaralkyl-; R₂ and R_(2′) are independently chosen from hydrogen,optionally substituted alkyl-, optionally substituted aryl-, optionallysubstituted aralkyl-, optionally substituted heteroaryl-, and optionallysubstituted heteroaralkyl-; or R₂ and R₂ taken together form anoptionally substituted 3- to 7-membered ring; R₁₂ is chosen fromhydrogen, optionally substituted alkyl-, optionally substituted aryl-,optionally substituted aralkyl-, optionally substituted heteroaryl-,optionally substituted heteroaralkyl-, —C(O)—R₃, and —S(O)₂—R_(3a); R₃is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-, R₁₅O—and R₁₇—NH—; R_(3a) is chosen from optionally substituted alkyl-,optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaryl-, optionally substitutedheteroaralkyl-, and R₁₇—NH—; R₄ is chosen from hydrogen, optionallysubstituted alkyl-, optionally substituted aryl-, optionally substitutedaralkyl-, optionally substituted heteroaralkyl-, and optionallysubstituted heterocyclyl-; or R₄ taken together with R₁₂, and thenitrogen to which they are bound, form an optionally substituted 5- to12-membered nitrogen-containing heterocycle, which optionallyincorporates one or two additional heteroatoms, selected from N, O, andS in the heterocycle ring; or R₄ taken together with R₂ form anoptionally substituted 5- to 12-membered nitrogen-containingheterocycle, which optionally incorporates one or two additionalheteroatoms, selected from N, O, and S in the heterocycle ring; R₅, R₆,R₇, and R₈ are independently chosen from hydrogen, acyl, optionallysubstituted alkyl-, optionally substituted alkoxy, halogen, hydroxyl,nitro, cyano, optionally substituted amino, alkylsulfonyl-,alkylsulfonamido-, alkylthio-, carboxyalkyl-, carboxamido-,aminocarbonyl-, optionally substituted aryl and optionally substitutedheteroaryl; R₁₅ is chosen from optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, and optionally substituted heteroaralkyl-; andR₁₇ is hydrogen, optionally substituted alkyl, optionally substitutedaryl, optionally substituted aralkyl, optionally substituted heteroaryl,or optionally substituted heteroaralkyl-.
 3. At least one chemicalentity chosen from compounds of Formula III:

and pharmaceutically acceptable salts, solvates, crystal forms,chelates, non-covalent complexes, prodrugs, and mixtures thereof,wherein R₁ is chosen from hydrogen, optionally substituted alkyl-,optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaryl-, and optionally substitutedheteroaralkyl-; R₃ is chosen from hydrogen, optionally substitutedalkyl-, optionally substituted aryl-, optionally substituted aralkyl-,optionally substituted heteroaryl-, optionally substitutedheteroaralkyl-, R₁₅O— and R₁₇—NH—; R_(3a) is chosen from optionallysubstituted alkyl-, optionally substituted aryl-, optionally substitutedaralkyl-, optionally substituted heteroaryl-, optionally substitutedheteroaralkyl-, and R₁₇—NH—; R₅, R₆, R₇, and R₈ are independently chosenfrom hydrogen, acyl, optionally substituted alkyl-, optionallysubstituted alkoxy, halogen, hydroxyl, nitro, cyano, optionallysubstituted amino, alkylsulfonyl-, alkylsulfonamido-, alkylthio-,carboxyalkyl-, carboxamido-, aminocarbonyl-, optionally substituted aryland optionally substituted heteroaryl-, provided that R₅, R₆, R₇ and R₈is absent where W, X, Y, or Z, respectively, is —N═, O, S or absent; R₁₅is chosen from optionally substituted alkyl-, optionally substitutedaryl-, optionally substituted aralkyl-, optionally substitutedheteroaryl-, and optionally substituted heteroaralkyl-; R₁₇ is hydrogen,optionally substituted alkyl, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted heteroaryl, or optionallysubstituted heteroaralkyl-; either R₂ and R_(2′) are independentlychosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, and optionally substituted heteroaralkyl- or R₂and R_(2′) taken together form an optionally substituted 3- to7-membered ring; and R₁₂ taken together with R₄, and the nitrogen towhich they are bound, form an optionally substituted 5- to 12-memberednitrogen-containing heterocycle, which optionally incorporates one ortwo additional heteroatoms, selected from N, O, and S in the heterocyclering, provided that such 5-membered nitrogen-containing heterocycle isnot an optionally substituted imidazolyl or imidazolinyl ring; or R₂taken together with R₄ form an optionally substituted 5- to 12-memberednitrogen-containing heterocycle, which optionally incorporates one ortwo additional heteroatoms, selected from N, O, and S in the heterocyclering; R_(2′) is hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, or optionally substituted heteroaralkyl; andR₁₂ is chosen from hydrogen, optionally substituted alkyl-, optionallysubstituted aryl-, optionally substituted aralkyl-, optionallysubstituted heteroaryl-, optionally substituted heteroaralkyl-,—C(O)—R₃, and —S(O)₂—R_(3a).
 4. At least one chemical entity of claim 1wherein R₁ is hydrogen, optionally substituted C₁-C₄ alkyl, optionallysubstituted phenyl-C₁-C₄-alkyl-, optionally substitutedheteroaryl-C₁-C₄-alkyl-, optionally substituted naphthalenylmethyl,optionally substituted phenyl, or naphthyl.
 5. At least one chemicalentity of claim 4 wherein R₁ is optionally substitutedphenyl-C₁-C₄-alkyl-, optionally substituted heteroaryl-C₁-C₄-alkyl-,optionally substituted naphthalenylmethyl, optionally substitutedphenyl, or naphthyl.
 6. At least one chemical entity of claim 5 whereinR₁ is naphthyl, phenyl, bromophenyl, chlorophenyl, methoxyphenyl,ethoxyphenyl, tolyl, dimethylphenyl, chorofluorophenyl,methylchlorophenyl, ethylphenyl, phenethyl, benzyl, halobenzyl,methylbenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl, dichlorobenzyl,dimethoxybenzyl, or naphthalenylmethyl.
 7. At least one chemical entityof claim 6 wherein R₁ is benzyl, halobenzyl, methylbenzyl,hydroxybenzyl, cyanobenzyl, methoxybenzyl, or naphthalenylmethyl.
 8. Atleast one chemical entity of claim 7 wherein R₁ is benzyl.
 9. At leastone chemical entity of claim 1 wherein, when present, R₅, R₆, R₇, and R₈are independently chosen from hydrogen, hydroxyl, halo, optionallysubstituted C₁-C₄ alkyl-, C₁-C₄ alkoxy, cyano, amino, substituted amino,or carbamyl-.
 10. At least one chemical entity of claim 9 wherein R₅,R₆, R₇, and R₈ are independently chosen form methoxy, methyl,trifluoromethyl, cyano, hydrogen and halo.
 11. At least one chemicalentity of claim 9 wherein R₅ is hydrogen or halo; R₆ is hydrogen,optionally substituted C₁-C₄ alkyl- or halo; R₇ is hydrogen, halo,optionally substituted C₁-C₄ alkyl-, C₁-C₄ alkoxy, cyano, substitutedamino, or carbamyl-; and R₈ is hydrogen, C₁-C₄ alkyl-, C₁-C₄ alkoxy,hydroxy, or halo.
 12. At least one chemical entity of claim 1 wherein R₂is optionally substituted C₁-C₄ alkyl-, and R_(2′) is hydrogen oroptionally substituted C₁-C₄ alkyl-.
 13. At least one chemical entity ofclaim 12 wherein R_(2′) is hydrogen and R₂ is optionally substitutedC₁-C₄ alkyl-.
 14. At least one chemical entity of claim 13 wherein R₂ ischosen from methyl-, ethyl-, propyl, butyl, methylthioethyl-,methylthiomethyl-, aminobutyl-, (CBZ)aminobutyl-, cyclohexylmethyl-,benzyloxymethyl-, methylsulfanylethyl-, methylsulfanylmethyl-, andhydroxymethyl.
 15. At least one chemical entity of claim 14 wherein R₂is ethyl or propyl.
 16. At least one chemical entity of claim 15 whereinR₂ is i-propyl.
 17. At least one chemical entity of claim 1 wherein R₄taken together with R₁₂ forms an optionally substituted diazepinonering.
 18. At least one chemical entity of claim 1 wherein R₄ takentogether with R₁₂ and the nitrogen to which they are bound, forms anoptionally substituted imidazolyl ring.
 19. At least one chemical entityof claim 1 wherein R₄ taken together with R₁₂ forms an optionallysubstituted imidazolinyl ring.
 20. At least one chemical entity of claim1 wherein R₄ is chosen from hydrogen, C₁-C₄ alkyl; cyclohexyl; phenylsubstituted with hydroxyl, C₁-C₄ alkoxy or C₁-C₄ alkyl; benzyl; andR₁₆-alkylene-, wherein R₁₆ is hydroxyl, carboxy, (C₁-C₄alkoxy)carbonyl-, di(C₁-C₄ alkyl)amino-, (C₁-C₄ alkyl)amino-, amino,(C₁-C₄ alkoxy)carbonylamino-, C₁-C₄ alkoxy-, or optionally substitutedN-heterocyclyl-.
 21. At least one chemical entity of claim 20 wherein R₄is chosen from hydrogen, methyl, ethyl, propyl, butyl, cyclohexyl,carboxyethyl, carboxymethyl, methoxyethyl, hydroxyethyl, hydroxypropyl,dimethylaminoethyl, dimethylaminopropyl, diethylaminoethyl,diethylaminopropyl, aminopropyl, methylaminopropyl,2,2-dimethyl-3-(dimethylamino)propyl, aminoethyl, aminobutyl,aminopentyl, aminohexyl, isopropylaminopropyl, diisopropylaminoethyl,1-methyl-4-(diethylamino)butyl, (t-Boc)aminopropyl, hydroxyphenyl,benzyl, methoxyphenyl, methylmethoxyphenyl, dimethylphenyl, tolyl,ethylphenyl, (oxopyrrolidinyl)propyl, (methoxycarbonyl)ethyl,benzylpiperidinyl, pyridinylethyl, pyridinylmethyl, morpholinylethylmorpholinylpropyl, piperidinyl, azetidinylmethyl, azetidinylethyl,azetidinylpropyl pyrrolidinylethyl, pyrrolidinylpropyl,piperidinylmethyl, piperidinylethyl, imidazolylpropyl, imidazolylethyl,(ethylpyrrolidinyl)methyl, (methylpyrrolidinyl)ethyl,(methylpiperidinyl)propyl, (methylpiperazinyl)propyl, furanylmethyl andindolylethyl.
 22. At least one chemical entity of claim 20 wherein R₄ isR₁₆-alkylene-, wherein R₁₆ is amino, C₁-C₄ alkylamino-, di(C₁-C₄alkyl)amino-, C₁-C₄ alkoxy-, hydroxyl, or N-heterocyclyl.
 23. At leastone chemical entity of claim 22 wherein R₁₆ is amino.
 24. At least onechemical entity of claim 22 wherein R₄ is aminoethyl, aminopropyl,aminobutyl, aminopentyl, aminohexyl, methylaminoethyl,methylaminopropyl, methylaminobutyl, methylaminopentyl,methylaminohexyl, dimethylaminoethyl, dimethylaminopropyl,dimethylaminobutyl, dimethylaminopentyl, dimethylaminohexyl,ethylaminoethyl, ethylaminopropyl, ethylaminobutyl, ethylaminopentyl,ethylaminohexyl, diethylaminoethyl, diethylaminopropyl,diethylaminobutyyl, diethylaminopentyl, or diethylaminohexyl.
 25. Atleast one chemical entity of claim 24 wherein R₄ is aminopropyl.
 26. Atleast one chemical entity of claim 1 wherein one of T and T′ is absentand the other is optionally substituted alkylene.
 27. At least onechemical entity of claim 1 wherein R₁₂ is optionally substituted C₁-C₁₃alkyl; optionally substituted aralkyl; and optionally substitutedheteroaralkyl.
 28. At least one chemical entity of claim 1 wherein R₁₂is —C(O)R₃ and R₃ is chosen from optionally substituted C₁-C₈ alkyl,optionally substituted aryl-C₁-C₄-alkyl-, optionally substitutedheteroaryl-C₁-C₄-alkyl-, optionally substituted heteroaryl, andoptionally substituted aryl.
 29. At least one chemical entity of claim28 wherein R₃ is chosen from phenyl; phenyl substituted with one or moreof the following substituents: halo; C₁-C₄ alkyl; C₁-C₄ alkylsubstituted with hydroxy; C₁-C₄ alkoxy; C₁-C₄ alkyl substituted withC₁-C₄ alkoxy, halo, nitro, formyl, carboxy, cyano, methylenedioxy,ethylenedioxy, acyl, —N-acyl or trifluoromethyl; benzyl; phenoxymethyl-;halophenoxymethyl-; phenylvinyl-; heteroaryl-; heteroaryl-substitutedwith C₁-C₄ alkyl or C₁-C₄ alkyl substituted with halo; C₁-C₄ alkylsubstituted with C₁-C₄ alkoxy-; and benzyloxymethyl-.
 30. At least onechemical entity of claim 29 wherein R₃ is chosen from phenyl,halophenyl, dihalophenyl, cyanophenyl, halo(trifluoromethyl)phenyl,hydroxymethylphenyl, methoxymethylphenyl, methoxyphenyl, ethoxyphenyl,carboxyphenyl, formylphenyl, ethylphenyl, tolyl, methylenedioxyphenyl,ethylenedioxyphenyl, methoxychlorophenyl, dihydro-benzodioxinyl,methylhalophenyl, trifluoromethylphenyl, furanyl, C₁-C₄ alkylsubstituted furanyl, trifluoromethylfuranyl, C₁-C₄ alkyl substitutedtrifluoromethylfuranyl, benzofuranyl, thiophenyl, C₁-C₄ alkylsubstituted thiophenyl, benzothiophenyl, benzothiadiazolyl, pyridinyl,indolyl, methylpyridinyl, trifluoromethylpyridinyl, pyrrolyl,quinolinyl, picolinyl, pyrazolyl, C₁-C₄ alkyl substituted pyrazolyl,N-methyl pyrazolyl, C₁-C₄ alkyl substituted N-methyl pyrazolyl, C₁-C₄alkyl substituted pyrazinyl, C₁-C₄ alkyl substituted isoxazolyl,benzoisoxazolyl, morpholinomethyl, methylthiomethyl, methoxymethyl,N-methyl imidazolyl, and imidazolyl.
 31. At least one chemical entity ofclaim 28 wherein R₃ is optionally substituted phenyl.
 32. At least onechemical entity of claim 31 wherein R₃ is tolyl, halophenyl,methylhalophenyl, hydroxymethylphenyl, halo(trifluoromethyl)phenyl-,methylenedioxyphenyl, formylphenyl or cyanophenyl.
 33. At least onechemical entity of claim 1 wherein R₁₂ is —SO₂R_(3a) and R_(3a) ischosen from C₁-C₁₃ alkyl-; phenyl-; naphthyl; phenyl substituted withcyano, halo, lower-alkyl-, lower-alkoxy, nitro, methylenedioxy, ortrifluoromethyl-; biphenylyl and heteroaryl.
 34. At least one chemicalentity of claim 1 wherein T and T′ are absent.
 35. At least one chemicalentity of claim 1 wherein one of W, X, Y, and Z is N and the others areC; R₁ is benzyl, halobenzyl, methoxybenzyl-, cyanobenzyl, ornaphthalenylmethyl-; R₂ is optionally substituted C₁-C₄ alkyl; R_(2′) ishydrogen; R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,amino, alkylamino, dialkylamino, hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy and cyano; T and T′ areindependently a covalent bond or optionally substituted lower alkylene;R₁₂ is —C(O)R₃ wherein R₃ is optionally substituted C₁-C₈ alkyl,optionally substituted heteroaryl, and optionally substituted aryl; andR₄ is R₁₆-alkylene-, wherein R₁₆ is amino, C₁-C₄ alkylamino-, di(C₁-C₄alkyl)amino-, C₁-C₄ alkoxy-, hydroxyl, or N-heterocyclyl.
 36. At leastone chemical entity of claim 35 wherein R₁ is benzyl, halobenzyl,methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; and R₂ is propyl. 37.At least one chemical entity of claim 1 wherein one of W, X, Y, and Z isN and the others are C; R₁ is benzyl, halobenzyl, methoxybenzyl,cyanobenzyl, or naphthalenylmethyl; R₂ is optionally substituted C₁-C₄alkyl; R₂ is hydrogen; R₅, R₆, R₇, and R₈ are independently chosen fromhydrogen, amino, alkylamino, dialkylamino, hydroxyl, halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy and cyano; T andT′ are independently a covalent bond or optionally substituted loweralkylene; and R₄ taken together with R₁₂ form an optionally substitutedimidazolyl.
 38. At least one chemical entity of claim 1 wherein one ofW, X, Y, and Z is N and the others are C; R₁ is benzyl, halobenzyl,methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; R₂ is optionallysubstituted C₁-C₄ alkyl; R_(2′) is hydrogen; R₅, R₆, R₇, and R₈ areindependently chosen from hydrogen, amino, alkylamino, dialkylamino,hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄haloalkoxy and cyano; T and T′ are independently a covalent bond oroptionally substituted lower alkylene; and R₄ taken together with R₁₂form an optionally substituted imidazolidinyl ring.
 39. At least onechemical entity of claim 1 wherein one of W, X, Y, and Z is N and theothers are C; R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, ornaphthalenylmethyl; R₂ is optionally substituted C₁-C₄ alkyl; R_(2′) ishydrogen; R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,amino, alkylamino, dialkylamino, hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy and cyano; T and T′ areindependently a covalent bond or optionally substituted lower alkylene;and R₄ taken together with R₁₂ form an optionally substitutedpiperazinyl ring.
 40. At least one chemical entity of claim 1 whereinone of W, X, Y, and Z is N and the others are C; R₁ is benzyl,halobenzyl, methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; R₂ isoptionally substituted C₁-C₄ alkyl; R_(2′) is hydrogen; R₅, R₆, R₇, andR₈ are independently chosen from hydrogen, amino, alkylamino,dialkylamino, hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄alkoxy, C₁-C₄ haloalkoxy and cyano; T and T′ are independently acovalent bond or optionally substituted lower alkylene; and R₄ takentogether with R₁₂ form an optionally substituted diazepinoyl ring. 41.At least one chemical entity of claim 2 wherein R₁ is benzyl,halobenzyl, methoxybenzyl-, cyanobenzyl, or naphthalenylmethyl-; R₂ isoptionally substituted C₁-C₄ alkyl; R_(2′) is hydrogen; R₅, R₆, R₇, andR₈ are independently chosen from hydrogen, amino, alkylamino,dialkylamino, hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄alkoxy, C₁-C₄ haloalkoxy and cyano; one of T and T′ is absent and theother is optionally substituted alkylene; R₁₂ is —C(O)R₃ wherein R₃ isoptionally substituted C₁-C₈ alkyl, optionally substituted heteroaryl,and optionally substituted aryl; and R₄ is R₁₆-alkylene-, wherein R₁₆ isamino, C₁-C₄ alkylamino-, di(C₁-C₄ alkyl)amino-, C₁-C₄ alkoxy-,hydroxyl, or N-heterocyclyl.
 42. At least one chemical entity of claim41 wherein R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, ornaphthalenylmethyl; and R₂ is propyl.
 43. At least one chemical entityof claim 2 wherein R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl,or naphthalenylmethyl; R₂ is optionally substituted C₁-C₄ alkyl; R_(2′)is hydrogen; R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,amino, alkylamino, dialkylamino, hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy and cyano; one of T and T′ isabsent and the other is optionally substituted alkylene; and R₄ takentogether with R₁₂ form an optionally substituted imidazolyl.
 44. Atleast one chemical entity of claim 2 wherein R₁ is benzyl, halobenzyl,methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; R₂ is optionallysubstituted C₁-C₄ alkyl; R_(2′) is hydrogen; R₅, R₆, R₇, and R₈ areindependently chosen from hydrogen, amino, alkylamino, dialkylamino,hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄haloalkoxy and cyano; one of T and T′ is absent and the other isoptionally substituted alkylene; and R₄ taken together with R₁₂ form anoptionally substituted imidazolidinyl ring.
 45. At least one chemicalentity of claim 2 wherein R₁ is benzyl, halobenzyl, methoxybenzyl,cyanobenzyl, or naphthalenylmethyl; R₂ is optionally substituted C₁-C₄alkyl; R_(2′) is hydrogen; R₅, R₆, R₇, and R₈ are independently chosenfrom hydrogen, amino, alkylamino, dialkylamino, hydroxyl, halogen, C₁-C₄alkyl, C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy and cyano; one ofT and T′ is absent and the other is optionally substituted alkylene; andR₄ taken together with R₁₂ form an optionally substituted piperazinylring.
 46. At least one chemical entity of claim 2 wherein R₁ is benzyl,halobenzyl, methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; R₂ isoptionally substituted C₁-C₄ alkyl; R_(2′) is hydrogen; R₅, R₆, R₇, andR₈ are independently chosen from hydrogen, amino, alkylamino,dialkylamino, hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄alkoxy, C₁-C₄ haloalkoxy and cyano; one of T and T′ is absent and theother is optionally substituted alkylene; and R₄ taken together with R₁₂form an optionally substituted diazepinoyl ring.
 47. At least onechemical entity of claim 3 wherein R₁ is benzyl, halobenzyl,methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; R₂ is optionallysubstituted C₁-C₄ alkyl; R_(2′) is hydrogen; R₅, R₆, R₇, and R₈ areindependently chosen from hydrogen, amino, alkylamino, dialkylamino,hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄haloalkoxy and cyano; and R₄ taken together with R₁₂ form an optionallysubstituted piperazinyl ring.
 48. At least one chemical entity of claim3 wherein R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, ornaphthalenylmethyl; R₂ is optionally substituted C₁-C₄ alkyl; R_(2′) ishydrogen; R₅, R₆, R₇, and R₈ are independently chosen from hydrogen,amino, alkylamino, dialkylamino, hydroxyl, halogen, C₁-C₄ alkyl, C₁-C₄haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy and cyano; and R₄ takentogether with R₁₂ form an optionally substituted diazepinoyl ring.
 49. Apharmaceutical composition comprising at least one chemical entity ofclaim 1, and at least one pharmaceutically acceptable excipient.
 50. Apharmaceutical composition according to claim 49, wherein saidcomposition further comprises a chemotherapeutic agent.
 51. A method ofinhibiting KSP comprising contacting KSP with an effective amount of atleast one chemical entity according to claim
 1. 52. A method fortreating a cellular proliferative disease comprising administering to asubject in need of such treatment at least one chemical entity accordingto claim
 1. 53. A method according to claim 52, wherein said cellularproliferative disease is selected from cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation.
 54. (canceled)55. (canceled)